Blood Component Separation and Use

[[p.1]]
Blood Component
Separation and Use
'11.1 BLOOD COMPONENT SEPARATION
History
The use. of blood' components washed red cell dates back to 1902 when Hedon used it in animal
experimentation with rabbit, Fleig's experimental plasma pherisis in rabbits in 1910 was followed by
Abel etal. in 1914.. It was Castellanos in 1937 who first transfused concentrated RBC in human being.
It was during second world war, between
1940~44
extensive trial with blood components and plasma
was taken up by Macquaide and Mollison 1940, Cooksey and Horwitz 1944 (Mollison, 1983). With
introduction of plastic equipment of transfusion by Walter and Murphy, 1952, the component separation
became easier' and it started developing rapidly. A new era of pheresis (plasmapheresis, cytapheresis,
leucopheresis and haemapheresis) was opened up in the field of transfusion practice and due to scarce
availability of human blood, a logical use of whole blood in the form of component started gaining
ground and was accepted by medical community as a strategy for optimum and effective utilisation of
blood.. A statistical data collected from advertising material of Bharat serums andvaccines (27 road,
wagle estate, Thane, Mumbai~4(0604)
cites. Areport submitted by Iguchi, Department of Surgery, Kyushu
University at 12th Japanese transfusion association general meeting (1963) states that a collection of
200 ml of blood per donor from 100 donors which when used as whole blood (stored) fulfilled the need
of 133 patient including 5% loss, average transfusion volume was 1425 ml per patient whereas the same
amount of collection whe., used as components (Fibrinogen, y-globulin, albumin, whole blood, plasma
protein, resuspended RBC). Can treat 679 patients suffering from different medical and surgical condition
viz. hypoprotenemia, measles, shock, post-operative resussicitation/recovery. Lung surgery, cardiac
surgery, anemias, abnormal obstetrical condition etc.
The Indian pharmacopiaalso recognises the use of bloodcomponents evidencedfrom the catagorisation
ofwhole human blood as blood replinisher, concentrated human RBC as blood replenisher, normal human
plasmaas plasmavolumerestorer, dried human plasmaas plasmavolumerestorerand humanplasmaprotein
fraction as blood volume supporter and laid down the minimum standards to be observed prior to their use
viz. sterility, assay (photo colorimetric haemoglobin estimation in case whole blood, RBC etc.) storage,
labelling, pyrogen, ABO and Rh grouping, tests for haemolysin etc. Fordetails consultDrugs and Cosmetics
Act, Part XII Band C. Also DGI:IS guidelines may be consulted in this respect.
322

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Blood Component Separation and Use
323
The whole human blood has two main components namely solid cellular component and liquid
component or plasma. For details refer to the Chapter of 2.1 and 2.2
The Physiochemical study reveals that different component of blood has different specific gravity
(Table11.1). This has been taken as an advantage in the blood component separation.The applicationof
this knowledge of physical property, a nearly static factor into dynamics of differential centrifugation
has resulted in effective separation of the components in the whole human blood.
These components can be separated from the whole blood by :
(i) Conventional technique or by
(ii) Cell separator
In conventionaltechniquethe refrigerated centrifuge machine is employed, centrifugation,freezing
and thawing are the process involved in the separation of blood component in vitro through a
semiautomatic operation.The speedof therotor and spinningtime arethe operationalvariablesconsidered
pivotal to the whole process and thecentrifuge should be calibrated at each operation to suit the needs of
particularcomponentto be prepared(accordingto instruction manual).The optimum speed and spintime
for the componentscan be calculated.The instruction manual mostly mentions the acceleration time and
speed necessary.
. As mentioned earlier the components are prepared by application of differential centrifugation at
different relative centrifugal force. This relative centrifugal force can be converted into rpm by the
following method where the radius of the rotor has to be taken into consideration.
(a) It can-becalculated by reference to the nomogram (Fig. 11.1).
(b) ReF in g -= 118 X 10-7 x R X N2where ro=radius in em
N = speed of rotation in rate per minute.
Table 11.1. Whole human blood (spec~fic
gravity : 1.055)
- I .
Solid component
(Cells)
I
L·
id
-I
iqui
component
(plasma)
sp. gravity 1.027
I
Leucocyte
poor, RBC
-I
Erythrocyte
(sp. gr. 1.095)
I~---'--'--"
Packed
RBC
Granulocyte
concentrate
Leucocyte
(sp. gr. 1.065)
Platelet
concentrate
I
Thrombocyte
(sp. gr. 1.032)
I
Plasma derivative
1. Albumin
2. PPF
3. AHF
4. Y globulin or
immunoglobulin
5. Fibrinogen
6. Coagulation factors
other than fibrinogen
Plasma component
1. FFP
2. Single donor plasma
3. Cryo precipitate
4. Cryo poor plasma

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324
Blood Banking and Transfusion
Fig. 11.1. Reference to Nomogram.
2000
4000
3000
rpm
2000
800
500
9
12
CoMo
9
CoM.
6
CoM.
Rotatingradius
in com. or inch
The calculation by reference to nomogram is
commonly practiced and easier method. In this
method, we know any two of the variables we can
find out the third. An illustration follows which is only
for purpose of description and not as per scale, say
we know the rotating radius = 9 cm (a distance of 9
cm from the centre of rotation) and speed 3000 rpm
and if we join this two point the ruler intersects the g
scale approximately at 800 g and vice versa i.e, we
know ReF and rotating radius we can find out the
speed at rpm.
Materials required can be divided in two groups :
A. Consumables: Articles are to be replenished
regularly for smooth and continuous
operation.
B. Equipments required for uninterrupted operation.
(a) Consumables: CPD or CPDA blood bags (double, tripple, quadriple - quad bags), transfer
packs, connector tubes card board boxes used as container for plasma packs, clips made·of
aluminium, sealer, cannistar made of aluminium for freezing pack, cutter, stripper etc.
(b) Equipment: Refrigerated centrifuge, blood bank refrigerator, electromechanical deep freezer
operating range of-40 to -70°C laminar flow, weighing scale, plasma expressor, the sealer,
.cutter, stripper frequently undergoes wear and tear and so included in consumables.
The automated blood processor also known as cell separator are the latest technology that allows
fullproof and safe separation of components that helps us in better patient care and donor care (by
returningthe unwanted constituent back irito circulation and retaining only the required component).
Cell separators are of'two types :
(a) CFC continuous flow centrifugation
(b) IFe intermittent flow cell separator and both differs in working principle,
In the former the operation is continuously maintained that means blood is drawn and returned in a
continuous flow and in the later the process is intermittent that means withdrawal and return is intermittent
and is through one venous access instead of two venous access as in CFC. Disadvantages commonly
encountered are large extracorporeal volume and lengthy operation time in IFC whereas the operation
with CFC is speedy and relatively less extracorporeal volume.
The operative principle is based on "Centrifugal separation of the component due to differential
density gradient. The system is an integrated combination of components ofboth hardware and software.
The hardware includes a centrifuge and pumps connected with electrical controls and software consists
of disposable pheresis sets and plastic bags for collection of components" - Bharucha (lac cit.).
Different types of cell separators are available in the maret and may be inducted for use according to
the suitability of the particular blood bank or hospital. Machines in common use for CFC are CS 3000;
ffiM, 2997 and also "viva" from Diedco. The IFC machine in common use are 'progress' from Diedco
'·and haemonetics 30 and V 50, haemonetics V 50 has been introduced very recently. Clinical application
and donor selection for pheresis is dealt in detail subsequently in a separate section.

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Blood Component Separation and Use
325
Component separation by refrigerated centrifuge
The required components are .separated from whole human blood collected from the donor.
Donor recruitment
Selection criteria of the donors are the same as that for replacement donors or voluntary donors with
only exception that the mandatory tests for blood is done ahead ofcollection in contrary to the replacement
donation where all the tests are done after collection. The other criteria which are to be particularly
followed for blood collection in connection with component preparation.
1. ABO and Rh compatibility test.
2. Preferably related donors of same family are choosen - easy availability, HLA compatibility.
3. Platelet should not be less than 1,50,OOO/cmm.
4. Test for C.M.V. is desirable.
5. No coagulation defect either congenital or acquired.
6. History of drug intake particularly of aspirin group.
7. Haemoglobin should be preferably 13.5 gn1% andlor above particularly· when cell separators
are used for component separation
A good antecubital vein is desirable to ensure a single prick phlebotony and continuous uninterrupted
flow of blood, to be collected in a amount proportionate to the anticoagulant present in the single pack
or primary plastic bag of a double, triple or a quad system. The satellite bags are devoid of anticoagulant
and connected to each other and primary plastic bag through integral tubing. This closed system of
collection in double bag or tripple bag ensures maintenance of sterility ofthe content during the operation
of component separation and subsequent storage or utilisation.
Minimum precautions, deemed essential during the procedure are :
(a) Ratio of collected blood and anticoagulant (CPD or CPDA) is to be maintained.
(b) Proper balance of opposing cups by putting in equal weight.
(c) Broad side of bag to face outer wall of centrifuge.
(d) Rubber discs for proper balancing.
(e) Time and speed are critical factors.
(0 Strictly follow instruction manual for smooth operation.
(g) Allow centrifuge to stop completely before opening lid do not stop the rotor by hand.
(h) Plastic overwears are desirable.
(i) Set the temperature as per requirement, prior to starting of centrifuge machine
G) Recheck by a second person ensures a correct operation.
Simplified account ofseparation ofdifferent component from whole human blood has been described
·through a flow chart diagram (Table 11.2) for an easy understanding of the procedure.
1. Red 'blood cell, concentrate (packed cell)
It consists of packed red cell after separation or removal of plasma from whole human blood either by
sedimentation or centrifugation. Rlasma is not completely removed but a little amount is left behind
which is necessary for optimal survival of red cells. Packed cell volume differs according to procedure
adopted by preparation :

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Blood Banking and Transfusion
Table 11.2
WHOLE HUMAN BLOOD
Refrigeratedcentrifuge
Leucocyte
Rich
Plasma
40XG
4°C
20mm
RBC
Concentrate
(a) 3000 XG
(b) 375 XG
2°C to 24°C
(a) 3 min
(b) 20 min
1000 XG
Inverted
spin by
keeping
bag in
inverted
position
ABC
Concentrate
5000 x G
"---
4°C --
~
5 min
Suspend
bag in
inverted
position
inal.V.
Stand
Plasma
Freeze
dried
Plasma
Dry
Freeze
ABC
·PRP
(a) 5000 XG
(b) 3000 XG
(a) 5 min
(b) 20 min
1400XG
Buffy
Coat
Contain-
ing Leuco-
cyte (gra-
nulocyte
concent-
rated)
Plasma
Leucocyte
Poor RBe
Concentrate
Collect
Leucocyte
poor red
cells in
satellite
bag by
releasing
seal
between
two bags
200 ml of
normal saline
added through
connectortube
Removeconnector
Washed Red Cells
Platelet
Concentrate
2000 XG
within 6 hrs of collection
To be processed
PlateletPoor
Plasma (P.P.P.)
5000 XG
4°C to SoC
5min
Thaw at
4°C in cold
circulating
water bath
Cryoprecipitate
5000XG
4°C
5 min
Freeze
Plasma
at - 70°C
.....----e-xp-r-es-s-e-d-o-u-t--~---------,
F.F.P.
Red Blood Cells

[[p.6]]
Fig. 11.2. Plasma expressor.
Fig. 11.3. Placing of blood bag in
plasma expressor.
Blood Component Separation and Use
327
(i) Sedimented cells have a PCV of 60 to 70% approximately.
(ii) Centrifuged cells have PCV around 80%.
Preparation
Sedimentation
Blood is collected in a single bag, kept overnight undisturbed in an upright position at 4°C the red cells
settle down and supernatant plasma is expressed out by plasma extractor and transferred into a transfer
pack. Express about 250 ml of plasma and not more from a 450 ml of blood collected in bag.
Centrifugation
Collect appropriate volume of blood in a double bag. Centrifuge for appropriate time and speed as per
instruction manual. Ifaimed for manufacture of packed red cell only; then commonly employed variable
are 4000XG for ten minutes, subject to variation as per rotating radius. So, it is always better to follow
instruction manual, bags with over wears made of plastic is
placed in the bucket and buckets are properly balanced before
the machine is started. The desired variables are fed into
electronic component of the machine. The temperature can be
at 4°C or at 20°C to 24°C depending on the state on which
blood was preserved. If blood was preserved at 4°C, then the
centrifuge is programmed for 4°C and if blood is preserved at
20°C to 24°C, then the machine is also programmed for 20°C
to 24°C.
The refrigerated centrifuge is now switched on, machine
stops functioning according to programme fed in. Donot use
hand to stop rotor and allow the rotor to come to complete
standstill before you open the lid. Remove units/bags carefully
from the bucket without any disturbance.
Now place the centrifuged bag or sedimented bag on
plasma expressor (Fig. 11.2) with label facing back so that
interface of red blood cell and plasma can be visualised
properly.
Bend the breakaway canula in the integral tubing
connecting the primary and satellite bag express at least 3/4th
of plasma (Fig. 11.3) into the satellite bag. Apply metal sealer
clip to stop the flow of plasma temporarily after 3/4th of plasma
hac; been transfered, clipper is fixed at least 8 to 10 em away
from the primary bag. Now with a heat sealer double seal the
tubing near the clip. Remove clip separate the primary bag
containing RBe concentrated and release the pressure plate of
the plasma expressor. Label the unit as RBC concentrated date
of manufacture, expiry,ABO, Rh group, clearance of mandatory
test, licence no. over the primary bag; similarly the plasma in

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Blood Banking and Transfusion
satellite, bag is also labelled. The original anticoagulant used is also to bementioned viz. CPDor CPDA.
Both the units are now sent for storage and preservation, primary bag are stored at 1°C to 6°C and during
transportation, it should not exceed temperature more than 10°C, of course for a short transportation
period. Plasma in satellite bag is to be stored as per requirement viz. FFP, single donor plasma etc., shelf
life of RBC concentrated is same as that of whole blood i.e., 35 days after date of collection of blood
from the donor provided it is stored at recommended temperature. If at any time during processing the
seal is broken or any open method, Le., aspiration of plasma etc., is adopted a deadline of transfusion
within 24 hrs. is to be followed.
The RBC concentrate preparation contains less of antibody and chemicals or biochemical substances
such as citrate, sodium, potassium, protein, ammonium etc.
Uses and indications
Symptomatic anemia, decrease volume of red cells in vivo. In certain cardiac condition or pregnancy,
where circulatory overload can create problem for the patient, refractory anemia.
2. Saline washed packed cell (SWPC)
There are packed red cells which are produced after repeated washing with 0.9% saline (physiologic
saline) either manually or mechanically.
SWPC is prepared from packed RBC (procedure described above).
To a unit of packed RBC, add 200 ml 0.9% saline through a connector which connects the saline
bottle with the bag of packed RBC. The connecting tube is transparent tube through which saline passed
under laminar flow, once the 200 ml of 0.9% saline is transferred into the bag containing packed RBC,
remove the connector tube from the saline bottle and is kept attached with bag with spike cover replaced.
Put the bag with packed RBC and saline into buckets of refrigerated centrifuge, The buckets are balanced
properly. The machine now programmed for 2000 xG for 5 minutes at 4°C. The saline and packed RBe
are mixed well before machine is started. Machine is switched on and once it stops the supernatant
saline is expressed or discarded as waste. Again fresh saline is added to the packed RBC left behind after
saline is removed, process is repeated again and in this way at least three washes are done every time
under asceptic condition so that sterility is maintained. After the final wash, add another 75 to 100 ml of
0.9%. saline and store this SWPC at 4°C. This SWPC has to be used within 24 hours of preparation.
Uses
Mainly in recipients who have febrile transfusion reaction. In multitransfused recipient for preventing
alloimmunisation with HLA.
3. Leucocyte poor red cell concentrate
These are the product which consist of packed RBC unit from where as least 70% of originalleucocytes
are removed. It can be produced by various method:
(a) Centrifuge the whole blood and remove the buffy coat.
(b) Centrifugation and filtration.
(c) Washing of red cell (described).
(d) Freeze and deglycerolize.
(e) Leucodepletion

[[p.8]]
Blood Component SeparatJon and Use
329
The centrifugation of whole blood and removal of buffycoat is done following the traditional inverted
spin or upright spin.
Inverted spin method includes positioning of a whole blood unit in an inverted position into the
bucket of centrifuge and the machine is programmed for 5000 x G for 5 minutes at 4°C. After the
centrifugation, the blood bag usually a double bag is preferred, is kept in an inverted position in a LV.
stand, the breakaway canula in the connecting tube is made bend and thus the connection is opened and
seal is broken, leucocyte poor red cell thus collects in satellite bag when an amount of at least 100 ml
(buffy coat) is left behind the connecting tube is double sealed and satellite bag is separated, satellite bag
labelled.
In upright spin the same procedure of centrifugation with same programme follows, the blood bags
are placed in upright position in the bucket. Following centrifugation the buffy coat alongwith at least
10 to 15 ml of top blood is also removed to ensure proper component separation. It may be siphoned off
or expressed in plasma expressor. Double seal the connecting tube and satellite bag removed and stored
at 4°C. Saline (0.9%) may be added to blood bag prior to centrifugation in upright spin method.
Shelflife of leucocyte poor R.B. concentrate is limited to 24 hrs. only and the yield contains leucocyte
less than 1 x 109 cell.
In centrifugation and filtration method, the packed red cell is filtered through commercially available
leucofilter in the market. The whole human blood centrifuged and filtered or filtration is done first
followed by centrifugation. The WBC gets attached to the leucofilter as the blood passes through filter.
The shelf life is 24 hours at 4°C and the yield contains leucocyte less than 0.5 x 109 cells ideal for
transfusion to persons suffering from repeated febrile transfusion reaction.
Freeze and deglycerolize
In this method the unit of packed RBC is frozen first by adding glycerol (cryoprotective) and then the
unit is placed at -70°C to -90°C with protective overwears in the mechanical freezer or it may be placed
ultralow freezing at -150°C to -196°C in liquid nitrogen. Before deglycerolization, the unit is brought
out and kept at 1°C to 4°C first, then at RT thereby gradually changing the environment and slow
thawing is induced. Finally, thawed at 37°C water bath. Now the RBC are deglycerolised by washing
and centrifuging. In this process, we loose leucocyte around 90 to 98% and platelets around 98 to 100%,
the RBC loss is minimal, substantial "amount of plasma is also lost during the process. These frozen and
"deglycerolised unit, when transfused, does not cause any febrile reaction. Although, it is the best method
to get rid of leucocytes but due to prohibitive cost, it is not commonly employed, the second best is
saline washing, by this method, we get rid of about 85% leucocytes and the third method is centrifugation
and removal of buffy coat which removes leucocyte around 75 to 80% use and indications are same as
washed red cell concentrate. Leucocyte poor RBC concentrate should be stored at 4°C for not more than
24 hours and are to be transfused within 24 hours.
leucodepletion
(a) Leucodepleted red cells can be obtained by a recently introduced procedure (ref. Chapter 3, 4).
(b) Integral filtration blood pack system : In this system multiple bag are connected with an online
leukodepletion filter. After collection of whole blood in the primary bag, the blood is allowed to
pass through the attached leukodepletion filter before further processing. J.eucocytes are trapped
in the filter Whenthe blood flows through the filter. The advantage in this method (i) Leucocytes

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330
Blood Banking and Transfusion
which are fresh are trapped in intact form and so there is less chance of release of cytokinase
which are thought to be responsible for fibrile reaction, and (ii) qualitative superiority, if compared
to bedside filtration.
From the leukodepleted blood, thus available for use, leukodepleted components can also be processed
as per requirement.
Commercially available in the market are Baxter Fenawel's equipment: (1) Pack pure WB system
(conventional whole blood leukodepletion system), (2) Optipure RC, optipress II and optilink together
provides the technology that relTIOVeS the dependence on single critical filtration procedure.
4. Platelet reach plasma
The component of plasma with raised concentration of platelet present in it. This is also known as PRP
in short form. This can also be prepared by centrifugation of whole human blood. The unit containing
whole human blood usually in a double or tripple bag is placed in the bucket of the centrifuge, within six
(6) hours of collection. The centrifuge cup bucket are properly balanced. The blood collected in a bag
containing anticoagulant (CPD/CPDA) in primary bag through a single venepuncture. Blood and
anticoagulant should be properly mixed and maintained at a temperature of 20°C to 24°C. Ensure the
donor has not taken any aspirin group of drug during past 72 hours or any decongestant and antihistamin.
The machine is now programmed for 3000 x G for 3 minutes at 20°C or 3750 for 20 minutes at
20
0e to 24°C. Once the centrifugation is complete as per programme. Now bring out the bag out of
bucket/cup and place it in plasma expressor, Express out approximately 4/5th of the plasma (platelet
reach) into the first satellite bag by bending the breakaway cannula in" the connecting tube between
primary and first satellite bag. Now the primary bag is separated from first satellite bag and the second,
third bags are allowed to remain together, so that third bag can be used for PPP (Platelet Poor Plasma),
if PC is to be manufactured. The second and third bag with PRP is preserved at a temperature of 20°C to
24°C.
To prepare platelet concentrate, a heavy spin is used is 5000 x G for 5 minutes or 3000 x G for 20
minutes at a temperature of 20°C to 24°C. After centrifugation is over, the two bags, i.e., second and
third bag of original is brought out from the cup of centrifuge and second bag is placed in plasma
expressor, the bending of breakaway cannula in the connector tube between second and third bag is
done and the supernatant PPP is expressed out in third bag and only 50 ml plasma with platelet is left
behind in the second bag. The bags are now labelled properly and preserved at 20
0e to 24°C, precisely
at 22°C. The bags containing platelet are allowed a period of rest for 60 minutes, after being brought out
from centrifuge. The bags are placed on a table top at RT and then at end of 60 minutes, preserved at
recommended temperature for 72 hours under constant agitation. The other bag containing PPP can be
used as single donor plasna and store accordingly or can be used commercially. The life of platelet can
be prolonged by maintaining the pH above 6.0 and the microenvironment inside the bag. A fall in pH
below 6.0 causes irreversible changes in shape, release of platelet granules and pseudopod formation
resulting in reduced in vivo survival of platelet. It has been observed that instead of usual PVC container,
ifbag, with polyolefin with no plasticiser (PL 732) andlorultra-thin-walled PVC with trimelliate plasticiser
(L 1240) bags are used as platelet container, then we have added advantage of increased permeability of
oxygen and CO2 which helps to maintain the desired microenvironment inside the container. Agitation
with platelet agitator enhances the gaseous exchange through membrane of the bag thereby maintains
pH and prevents aggregation of platelet. Flat bed agitator which are capable of strokes @ 70 cpm (cycle

[[p.10]]
Blood Component Separation and Use
331
per minute) at a temperature of 22°e has been found more suitable for the purpose. If the temperature is
fluctuating, then the help of platelet incubator is taken which maintains the desired environment of 200e
to 24°C. All this additional measure, if taken the platelet viability and function can be increased upto
seven days.
Platelet can also be stored at 1°C to 6°C without agitation hut better function and viability is observed
with platelet stored at 20°C to 24°C.
Some precautionary measure, if adopted during platelet processing needs attention viz. chilling
avoided during manufacturing process, donors must not take aspirin, the shelf life though extended upto
seven days by improvised method of storage transfusion of platelet should be done within five days to
avoid risk bacterial contamination, ABO and Rh compatibility is essential, HLAmatching is desirable.
The platelet processed by cell separator has been yielded with less alloantibody formation than that
manufactured by refrigerated centrifuge.
An unit of whole human blood of 450 ml that produces 50 ml of platelet concentrate should have a
yield of.
Platelet: 5.5 x 1010 platelets in at least 75% of units tested.
Plasma volume
(i) 40 to 70 ml, mean being 50 ml storage at 22°C with agitation.
(ii) 20 to 30 ml, if stored at 1°C to 6°C without agitation.
(iii) WBC - 108, RBC - trace.
(iv) pH - not less than 6.0 at the end of storage period.
Platelet yield can be calculated as follows :
In PRP : Platelets per cubic millimeter x 1000 x volume of PRP (in ml).
In PC : Platelet per cubic millimeter x ]000 x volume of PC (in ml).
Platelet transfusion is done through normal blood filter and microaggregate filters are not used to
prevent loss of platelet. ABO and Rh compatibility is essential to prevent haemolytic transfusion reaction
that may occur due to chance presence of red cells in PRPIPC. HLA matching though not essential but
desirable to avoid alloimmunisation in recipients with HLA antibody.
Platelet recovery in vivo is influenced by so many factors":
(a) Freshness of platelet.
(b)
10% decrease in recovery with every 24 hours of storage.
(c) Fever and infection.
(d) Secondary coaguJopathy - DIC.
(e)
Recovery may also be affected by anti-platelet antibody like anti-PLAl, others are rarely available
in general population but if present (anti-Kfr', anti-Duzo and anti-Bak) may cause lysis of
platelet.
The anticipated platelet increment can be calculated as follows :
Total No. of platelet transfused
x 2/3
Anticipated platelet increment = Blood volume/body weight in gms

[[p.11]]
332
Blood Banking and Transfusion
Method is yet to be developed for platelet matching but the commonly followed guideline is A or
AB platelet to A or AB recipients and '0' group recipient can receive any platelet. The same group
platelet is recommended for 'B' group recipient. The criteria for RhIT in females are same as that for
whole blood transfusion.
Single donor platelet is always to be preferred but in abscence, pooled platelet from random donors
can also be used. All the donors must be cleared for all mandatory tests.
There are different type ofplatelet preparation like pooled platelet, washed platelet, irradiated platelet,
frozen platelet, single donor platelet etc. and use is dictated by the need ofthe patient or as adjudged by
treating doctor. Mainly used for bleeding in thrombocytopenia or platelet function abnormality.
5. GranUlocyte concentrate
The buffy coat containing leucocyte can be prepared by refrigerated centrifuge from a single donor by
cell separator. A unit of whole human blood on centrifugation produces a buffy coat rich in white cells is
seen at the interface of red blood cells and supernatant plasma. This buffy coat contains about 50% of
white cell contaminated with red cell and thrombocytes, The specific gravity of RBC and granulocyte is
nearly similar so it is very difficult to separate the RBC from granulocyte by centrifugation only and as
such the buffy coat is contaminated with red cells.
Preparation
For preparation of granulocyte concentrate collected unit of blood is stored at 4°C for 4 (four) hours
only.Jt has to pass all mandatory tests and ABO, Rh compatibility before being processed for granulocyte
concentrate. The blood bags are kept. in the buckets/cups of the centrifuge according to description
given earlier, and centrifuged at 4°C appropriate speed and time is programmed into the machine, a light
spin is preferred (see table of component separation). Supernatant plasma is now expressed into first
satellite bag, the complete plasma portion is not removed at least 15 to.20 ml of plasma is left behind in
the primary bag. Plasma in first satellite bag can be used as single donor plasma. Now allow-a rest period
for the primary bag till the disturbance caused due to expression of plasma is settled. Once the plasma is
settled, place the primary bag into the plasma expressor again and express the residual plasma along
with 15 to 20 ml of top portion of the cell layer into transfer pack or another satellite bag. After the
process of expressing buffy coat is completed the primary bag is now separated from transfer pack of
satellite bag.
The yield consists of white cells, platelet and red cell approximately half the yield of white cells are
granulocyte and it differs from unit to unit and requires many unit to reach the desired label for granulocytes
which is approximately 1 x 1010•
.
The yield of granulocyte may be increased by prior administration of steroid-prednisolone to the
prospective donor; the granulocyte count reaches peak by 4 to 5 hours of administration. Red cell
sedimenting by rouleoux forming agent like dextran and hydroxyethyl starch helps to separate red cells
from granulocyte. So anyone of these or combination of two will give a better yield, granulocytes can
.also be processed by cell separator or by filtration leucapheresis or gravity leucapheresis described
under appropriate heading.
The granulocytes are used mainly in cases of scepticimia (gram negative scepticimia) where
neutropenia predominates the picture. In radiotherapy or chemotherapy also neutropeinia is commonly
observed. The granulocyte transfusion, in such cases plays a supportive role to antibiotic therapy viz.

[[p.12]]
Blood Component Separation and Use
333
scepticimia or supportive role to chemo radiotherapy. Desired label considered effective is 1 x 1010
granulocytes per day. This label of granulocyte in one unit can only be achieved by processing through
cell separator. The buffy coat prepared by centrifugation approximately consists of 0.5 to 0.6 x 109
granulocytes only and as such huge amount of buffy coat will be required to reach the desired label as
described above. Recovery of granulocyte in vivo is about 50% at the end of first hour after transfusion.
This is far less in HLA unmatched recipient or in multi-transfused cases probably due to presence of
HLA antibody.
The granulocyte concentrate is administered through blood administration set @ 5 x 109 granulocytes
per hour. Since there are many untoward effects that may occur due to granulocyte transfusion viz.
alJoimmunisation, GVHD, febrile reaction and pulmonary infiltration etc., it is always preferable to
transfuse irradiated granulocytes whichis preceded by administration of anti-histaminics and or steroids.
The recipient is monitored periodically to know the degree of improvement (clinically and evaluation of
laboratory parameters) and as the improvement reaches the desired label the transfusion is discontinued.
Normally four to six units of granulocyte concentrate is required to combat severe gram negative
neutropenia. Since the preparation of granulocyte concentrate IS costly, time consuming process and
with the advent of antibiotics scepticimia is no more a problem, the production ofgranulocyte concentrate
has become restricted and unless clearly indicated and prescribed, its production should not be ventured
into.
Stem cell
Stem cells are pluripotential and unspecialised cells in its primary stage of development. They are the
progenitors of the pools of cell can be termed as building block for blood (refer Chapter 2.2), immune
cells etc. Stem cells are found in :
(a) Bone marrow
(b) Cord blood
(c) Peripheral blood
Previously stem cells used to be collected: from bone marrow but now-a-days embryonic stem cells
are collected from cord blood. Cord blood stem cells are preferred more because ofeasyness ofcollection.
lower incidence of GVHD and infections from CMV etc.
The cord blood is a reach source of stem cell and can be collected from umbilical cord which
remains attached to the placenta after the delivery of child. The blood is collected in a specially designed
bag after obtaining consent from the mother. Blood is then screened for HIV, Hey, HBV, CMV, EBV,
syphillis, malaria. In addition to this HLA typing for class 1 and 2 antigens are also done. Blood is then
further processed for enriching the stem cell content, which·is then stored in a cryopreserved form..
The peripheral blood stem cells are collected by a closed method through cell separator machine
which is then stored in a cryopreserved form,
The peripheral blood stem cells are collected by a closed method through cell separator machine -
amicus MNC (mononuclear cells) manufactured by Baxter International.
Indication and use of stem cells :
(a) Embryonic stem cells from cord blood :"
(i) Thallasemia
(ii) Anemia: Fanconi's, sickle cell anemia

[[p.13]]
334
Blood Banking and Transfusion
(iii) Malignancy:
Lymphoma: Hodgekins, non-Hodgekins
Leukemia: AML, ALL, CML etc.
(b) Peripheral blood stem cell (PBSC) :
(i) Autologus transplant
(ii) Chemoradiotherapy
Plasma is available as single donor plasma, fresh fozen plasma (FFP), pooled plasma and lyophilised
pooled plasma..
The single donor plasma and FFP can be prepared by refrigerated centrifuge also a small scale
pooling of two to three units of plasma can be done after separation of plasma. But the production of
pooled plasma and lyophilised pooled plasma needs bigger establishment with sophisticated machinery
as these two products are manufactured on commercial basis. In India Haffkine Biopharmaceutical
Corporation Limited manufactures pooled plasma and lyophilised pooled plasma. In lyophilised pooled
plasma at least 10 donors plasma are pooled together in smaller scale and/or pooling ofrecovered plasma,
unutilised plasma that has reached expiry date.
Lyophilisation is a process by which we preserve the biological material by successively freezing
and drying in vacuum. Thus in lyophilised pooled plasma the biological value and property of plasma is
retained. This has to be reconstituted with 250 ml of wat~r
for injection before use.
In large scale manufacture, plasma are pooled in a plasma station and subsequently defibrinated,
thorough mixing is done, after which they are filtered and dispensed in glass containers which are
lyophilised and sealed under nitrogen.
Advantage
Extended shelf life, easy transport, can be used when plasma is not readily available in emergency.
Disadvantage
It has to be reconstituted before use, since they are pooled, chances of spread of hepatitis and AIDS
looms large, if proper precautionary measures are not followed.
Single donor frozen plasma
This is the plasma obtained from whole blood after six hours of collection but within a period of 72
hours and preserved in a frozen state. Processing of plasma after this period may be fraught with diminution
of coagulation factor particularly factor VIII and change in electrolyte and plasma pH.
Single unit of whole human blood preferably collected in a double bag and which was preserved at
4°C is selected for the purpose of preparation of single donor frozen plasma. Machine is programmed
for 5000 x G for 5 minutes at 4°C and the blood bag is placed in the bucket of centrifuge. Balancing of
opposing cups are carried on, lid is closed and machine switched on. At the end of centrifugation, the
bag is brought without disturbing the red cell layer, the bag is now placed in plasma expressor and
express 3/4th of plasma into the satellite bag after bending the break-away canula. Double seal the

[[p.14]]
Blood Component Separation and Use
335
tubing and separate from primary bag as described earlier. This plasma, if stored below 30°C can be
used up to 4 years as single donor plasma.
Use and indications are :
(a) Volume expanders in hypovolimia in shock, severe haemorrhage.
(b) Hypovolimia and hypoprotinimia - burns.
(c) Deficiency of stable coagulation factors.
(d) Albumin replacement, if FFP or PPF not readily available.
Fresh frozen plasma
This is the plasma that is obtained from whole blood within six hours of collection and is quickly freezed
and stored in a frozen state below 30°C.
Single unit of a whole human blood collected by single, clean (under asceptic condition) phlebotomy
with uninterrupted rapid flow of blood is collected in a double bag. An amount of 350 ml blood should
not take more than eight minutes for collection, thorough mixing of anticoagulant is a must. This blood
is to be stored at 4°C and has to be processed within six hours of collection. Place the bags within bucket
cup of refrigerated centrifuge and balance the opposite cups by putting an equal weight. Machine is
programmed for 5000 G,for 5 minutes at 2°C to 6°C. Once the operation is over the bags are brought out
without disturbing the red cell layer to avoid risk of contamination with red cell. Put the bags in plasma
expressorwith unlabelled side of bag facing operator to enable the operator a proper view of cell plasma
interface. Express out at least 3/4th of supernatant plasma into the satellite bag after bending the break-
away canula. Once transfer of plasma is over, clipping is done at 8 to 10 em distance from primary bag
with a metal clip for temporary stopping of plasma flow. Now double seal the tubing with a dielectric
sealer and separate the satellite bag. Label the plasma in the satellite bag. This bag is now rapidly frozen
after placing a tight rubber band around the bag and then keeping the bag with a plastic overwear as
follows:
(i) Bags laid flat in a electromechanical freezer at 70°C.
(ii) It can also be placed in a mixture of ethanol dry ice bath at 70°C.
(iii) Harris blast freezer.
.Rapid freezing recommended within 4 hours of blood collection for a better yield of coagulation
factors. The freezing is confirmed when the contents become stony hard. Some instacool freezer are
available at market that takes about 35 to 40 minutes and operates at -25°C, these instacool freezer are
improvised electromechanical freezer which are considered better than the freezers available in recent
past.
After freezing is over, ·it is stored at -30°C or below and it has been shown that most of the labile
coagulation factors can be preserved at this temperature for a period of one year. As such the FFP has a
life of one year at this temperature and after this same unit can be used as single donor plasma for
another 4 years. The .storing is done in an upright position of bag after removal of rubber band placed
before freezing. Transfusion of any plasma is not without hazard and as such group specific compatible
plasma is advised because contamination ofplasma with RBC is an accepted fact and is to be administered
after, thawing through standard administration set for blood, rate adjusted between 8 to 12 mVminute the
thawing done at 37°C in water bath keeping care. of the container membrane so that there is no leak
(previously or during thawing. The disappearance of indentation caused by rubber band during freezing

[[p.15]]
336
Blood Banking and Transfusion
indicates thawing has occurred. Thawing is also done as quick as possible under constant agitation, bag
should be removed the moment disappearance of rubber band indentation occurs. This is essential to
prevent formation of cryoprecipitate.
Thawed FFP is preserved at 100eand not to be preserved for a longer period. Recommended period
is 2 to 2~
hours during which it has to be transfused. It is not to refroozen and if not utilised, the FFP
label is removed and label for "recovered plasma" is fixed then despatch for commercial use.
The FFP which is 250 ml by volume contains 175 to 250 units of all labile and stable coagulation
factors viz. VIIi, IX, XI are 'the most important. Factor V, VII, XIII are found in lesser amount. It also
contains fibrinogen (approximately 400 mgm).
Uses and indications for FFP :
(a) Haemophilia - A, Band C
(b) DIe
(c) Inherited deficiency of factor V
(d) Deficiency of factors, VII, IX, X, XIII and II
(e) Deficiency of fibrinogen - afibrinogenemia and hypofibrinogenemia
(f)
As supportive measure to vitamin K therapy
(g) Sometimes during bypass operation particularly cardiopulmonary bypass - how it works is not
yet clear.
Cryoprecipitate
They are white gelatinous material or precipitated plasma proteins and are reach source of factor Vlll,
VWF and fibrinogen, processed from an FFP unit.
Blood is at first collected in a tripple bag through a single attempt clean phlebotomy and FFP is
made which is freezed and stored after separating the primary bag only.
To manufacture cryoprecipitate, the FFP unit has to be thawed at 4°C in cold circulating water bath.
This thawed FFP is now centrifuged at 5000 XG for five minutes, at 4°C. The centrifuged unit is carefully
removed from the bucket without disturbing the layers and placed in a plasma expressor. The supernatant
plasma is expressed into third satellite bag. The second and third satellite bags are separated and double
sealed. Second satellite bag contains CPPT and third satellite bag contains cryopoor plasma which can
be used as single donor plasma.
It can also be processed by hanging the FFP unit from a hook upside down at a temperature of 4°C
for a period of 18 hours. From the second satellite bag with its parts down the factors vm deficient
plasma slowly flows down into' the third satellite bag - cryopoor plasma, what remains in the second
satellite (original) bag is about 15 to 20 ml ofCPPT - cold insoluble portion of plasma. The essentials in
this procedure are free air circulation around the bags maintained at temperature range of 2°C to 6°C
(possible in a refrigerator or walk-in cooler). If procedure is done inside a walk-in cooler, air circulation
is no problem but if it is done in a refrigerator, the air circulation is better ensured by observing both the
bags at half an hour interval by opening the refrigerator door. Thawing isdone at 4°C in cold circulating
water bath and can be done as follows. Place the FFP unit with rubber band around it in the cryo-
compression racks in the holder, then placed in a cold circulating water bath operating between 2°C to
6°C preferably 4°C, record temperature of water bath and the time when the FFP units are placed in

[[p.16]]
80 to 120 units
High level.
High level
20 to 25% of original level
150 to 250 mg
55mg
In lesser amount
Blood Component Separation and Use
337
water bath, units should be checked frequently for ice and slushy appearance gradually occurs in thawed
units and also disappearanceof indentation of rubber band. Complete thawing may even require upto 9()
minutes. Thawed plasma can now be put inside plastic overwear or plastic centrifuge bag ready fOT
immediate centrifugation 4"C.
Several factors influences the yield of CPPT and needs consideration for a better yield.
.(a)' Single attempt clean phelbotomy allowing free or uninterrupted flow of blood collected within
8 to 10 minutes in a tripple bag ensuring thorough mixing of blood and anticoagulant.
(b) Rapid freezin.g within 4 hours of collection and preserved at a temperature of -30°C or below,
of the FFP unit processed.
(c) Blood group belonging to A possess more than 8%of factorvm than that of '0' group persons.
(d) Injection of DDAVPthrough I.V. route, 15 minutes before donation has shown to increase the
yield of factor VIII in plasma of such donors.
(e) Plasma and heparinised plasma also shows a better yield on processing for factor VIII.
(0 Less old theFFP,better is the yield 'If factor VIII, there is gradual loss of factor VIII activity in
plasma stored at -30°C.
The cryoprecipitate can be preserved upto one year from the date of collection of blood, if the
processed CPPT is freezed within 8 hours of production and then preserved at or below -20°C.
Cryoprecipitate or cryoreach plasma has a volume of 15 to 20 ml approximately and contains the
following:
VIlle
VillVWF
VIII R-Ag
XllI
.Fibrinogen
Fibronectin
II, V and IX
Cryoglobulin
Uses and indications of cryoprecipitate (CPPf) is varied and it can be used in treatment of haemophilia,
Vonwillebrand'sdisease,Ole, deficiencyof factor XIII and fibrinogen deficiency (congenital or acquired
partial or complete).
Dosafle
One unit of CPPT.approximately raises the factor VIII by 10%.
Cell separators
Different type of cell separator available in the market and basic technology has already been described
and according to that there can be two types of technology. Intermittent (IFC) and continuous (CFC)
operation in a cell separator machine. The function (cell separation) and goal (process particular
component) are same but the mode of operation is different in intermittent flow cell separator and
continuous flow cell separator.The operative principle in both the machine is that venous bloodobtained
from peripheral vein of donor is being anticoagulatedby automaticmixing of proportionateanticoagulant

[[p.17]]
338
Blood Banking and Transfusion
and processed through differential centrifugation (centrifugal separation ofcomponents due to differential
density gradient), in the automatically operated centrifuge bowl/container, programmed to retain the
desired component and reinfusing back the rest to the donor, under a biologically closed system in
toxicologically safe material.
The intermittent flow centrifugation that operates on a discontinuous process was originally developed
by Tullis in sixties. A simplified system is a combination of centrifuge and two pumps, three way clamps,
holders for solution container etc. It is operated automatically through electrical control where
anticoagulant is uninterruptedly and thoroughly mixed in a proportionate ratio by means of two
interconnected pumps - one pumps in the anticoagulant and the other anticoagulates the incoming blood.
The speed of rotation and flow ratio is influenced by sizes of pump tubing. They operate whenever the
blood is being pumped into the centrifuge bowl. The blood is being drawn in successive batches and
separated into components. First the plasma flows out of effluent tube next in order is platelet followed
by white blood cell. After these are separated, red blood cells remains in the bowl. The desired component
as per programme (fed in) is harvested and rest is reinfused back into the donor.
Continuous flow centrifugation was also conceived and designed by Freirich and Judson around
sixties. The anticoagulated blood uninterruptedly flows into the separation container where it is separated
in accordance to the principle of differential centrifugation into component viz. red cells and plasma.
The plasma is PRP with or without leucocytes. Leucocytes are found in buffy coat. These separated
component of plasma are now directed to collection container where WBC and platelet are separated
and desired component is harvested, the uncollected part is removed by peristaltic pump, recombined
and reinfused back into the same donor, the extra corporeal volume limited to 5000 ml consist of volume
in circuit plus the volume collected. Almost all except the volume of desired component is reinfused
back into the donor. For details the accompanying literature with the machine may be consulted. The
recently developed CFC are more efficient and more sophisticated in technology and ensures proper
harvesting and maximal reinfusion with lesser extracorporeal volume under computerised guidance
through two venous accesses, one for incoming blood and other for returning blood, every procedure
has its own advantage and disadvantage (Table 11.3). IFC and CFC are no exception. So choice of
technique or machine has to be done after considering the advantage, disadvantage, economic status (of
user), infrastructure viz. trained personnel, building, equipment, various component demand and supply
required.
Donor criteria are almost same as selection of donor for whole human blood. The donors are tested
and cleared for all mandatory test. Haemoglobin not less than 13.5 gm%. Donor criteria varies according
to type of pheresis descril-v' with appropriate type of pheresis.
The effect on donors after pheresis procedure are :
(a) Fall in haemoglobin due to haemodilution, so repeat pheresis is not done immediately if the
haemoglobin is not within prescribed limit.
(b) Fall in platelet count and repeat pheresis withheld till platelet count is above 150 x l09nitre.
(c) Vasovagal attack may occur.
(d) Citrate toxicity : Paresthesia, chilling, may or may not be associated with increased stiffness of
muscle, nausea and vomitting.
(e) During reinfusion, patient may feel cold sometimes, now-a-days new machines are fitted with
blood warming device to warmup the reinfused blood.

[[p.18]]
Blood Component Separation and Use
339
Table. 11.3. Comparison of cell separators
Provisions
Intermittentflow cell separation
Continuous flow cell separator
Advantage
(a)
Safe and simple procedure
(a)
Speedy operation
(b) One venous access
(b) Extracorporeal volumeis comparativelyless
(c)
Easily transportable
(c) Comparatively bet~er
plasma pheresis
(d) Yieldof platelet and granulocyteis
comparatively better
(e) Comparatively cheaper
Disadvantage
(a) Slow operation
(a) Double venous access
(b) Extracorpoal volume is comparatively
(b)
Yieldof platelet and granulocyteis compa-
more
tively lower
(c)
Blood volume of donor'changes quickly
and also intermittently
Uses
(i) Apheresis (Pheresis) of platelet, plasma, white cells.
(ii) Plasma"exchange for removal of some undeserved element from our body and also for treating
specific diseases.
(iii) Therapeutic leucapheresis and platelet apheresis.
(iv) Red cell exchange.
11.2 APHARESIS
Apharesis is a procedure that involves collection of whole human blood from a donor that is duly
anticoagulated and then separated into different components, desired component is retained and rest
reinfused back to the same donor. It is essential to have a healthy, fit donor suitable to the requirement
(special donor criteria according to component is described later).
With the advance in technology clinical application of this procedure was tried and in soine cases
favourable results were obtained in removal of undeserved and harmful elements, components from the
body and also in treatment of some specific diseases. It has added advantage of diminished chance of
.alloimmunisation and reduced risk of TID.
So, there are two distinct uses of apharesis namely component separation and therapeutic
haemapheresis and they are carried out by cell separators, intermittent flow cell separator and continuous
flow cell separator (both described in the previous section).
Component separation by cell separators are mainly:
(i) Cytarpheresis i.e. separation of leucocytes and platelet
(ii) Plasmapheresis
(iii) Separation of neocytes.
Cytapheresis
This is employed mainly for collection of granulocytes and platelets.

[[p.19]]
340
Blood Banking and Transfusion
Donor criteria is same as that for whole blood transfusion but certain special criterias are set in for
donors undergoing pheresis.
(a) Donors written consent must be obtained and the whole procedure is explained to him so that he
can spend the time required for the whole operation and ready to face the untoward effects
relating to citrate toxicity and extracorporeal volume which occurs seldom.
(b) Platelet count should not be less than 150 x 109/1and donors should not take aspirin group of
drug.
(c) Haemoglobin 13.5 gm%.
(d) Granulocyte count 4 x 109/1and above gives a better yield.
(e) HLA matched donor is desirable but-not essential.
(0 Donors must be cleared for all mandatory test (VDRL, malaria, HbSAg, HCV and HIV) and
test for CMV is desirable before being selected for donation.
(g) ABO and Rh matched donor is essential.
Granulocyte collection by cell separator is not without problem because of nearly similar specific
gravity of red cells and granulocyte, so differential centrifugation in the centrifuge bowl is not cleareut.
This results in heavy contamination of leucocytes with red cells.
Granulocyte components are clinically applied in the treatment of granulocytopenia. As such a
better yield of granulocyte is always desirable from a single operation to avoid frequent pheresis.
Corticosteroids (prednisolone or dexamethaoone) are known increase the granulocyte count probably
through release of granulocyte by bone marrow.
As such predonation administration ofcorticosteroid @ dose of 20 mgm orally according to accepted
protocol raises the yield of granulocyte.
Augmentation of granulocyte collection can be helped by administration of red cell sedimenting
agent or rouleaux forming agent like hydroxyethyl starch (HES) or Dextran to the input line of separator.
This enhances the sedimentation and rouleaux formation of red cells thereby better separation of buffy
coat (granulocytes, other leucocytes, platelets) with minimal contamination of red cells.
Anyone or combination ofboth will thus definitely increase the granulocyte count yield ofgranulocyte
harvested by cell separators.
Approximate yield of granulocyte harvested by cell separator is around 0.5-1 x 101°/200 to 400 mI
of plasma.
Uses
Granulocyte transfusion are mainly used as a supportive therapy in severe gram negative scepticimia
resistant to antibiotics and in cases of refractory. granulocytopenia.
Platelet collection by cell separator
Platelet collection by cell separator machine· is always considered better than refrigerated centrifuge
because of high yield and minimal platelet alloantibody formation: the yield of platelet again differs
with different type of cell separator. The CFC machines, travenoI CS 3000 and dual stage channel IBM
2997 gives a comparatively low yield with less leucocyte and almost nil red cell contamination whereas
IFC haemonatic 309 gives a high yield with moderately high leucocyte and appreciable, red cell

[[p.20]]
Blood Component Separation and Use
341
contamination. The recently introduced haemonetics V50 with a surge pump is considered,best for high
platelet yield with minimal of leucocyte and red blood cell contamination.
Platelet collection by model 30s haemonetics both single or double venous access can be used,
latter is preferred because of reduced cycle tirne and more acceptable to donor. In this system, blood is at
first pumped into the centrifuge bowl approximately @ 60 to 100 ml minute, the flow is reduced to 40
ml/minutes as soon as platelet appears at the shoulder of bowl after the plasma flows out through effluent
tube and collection of platelet starts only when platelet band progresses 4 Clll along the shoulder of the
centrifuge bowl, Collection continues for another 40 to 45 seconds and the flow rate blood during
collection period is maintained around 20 ml/min. Six cycle of operation are usually carried out to have
an appreciable yield of platelet.
Platelet viability and function is influenced by storage temperature and containers already described
in previous section and accordingly storage temperature, containers are to be recommended for a minimal
5 ~ys
storage. More over certain drugs viz. aspirin and related compound has an adverse effect on
platelet function and so selected donor must not take these drugs at least within 72hours prior to donation.
The ideal donor is of course ABO, Rh and HLA matched donor. To avoid HLA matching sometimes
nearest (blood related) kin is selected for donation purpose. Donor must be cleared for all mandatory test
and CMV.clearance is also desirable.
Platelet transfusion
Platelets are administered through a standard blood administration set fitted with filters. Platelets
manufactured from a unit of whole blood raises the platelet count by 10,000 to 12,OOO/min3 but the
platelet separated by cell separators has a yield better in comparison to that separated by refrigerated
centrifuge. The increment, therefore, is more than double with plaetlet concentrate separated by cell
separators and as such preferred for transfusion, ABO Rh compatible platelets are essential, HLA matched
platelets are desirable, particularly in multi-transfused cases. Rh grouping is done as precautionary measure
to avoid haemolytic transfusion reaction in red cell contamination and isoimmunisation in Rh negative
cases. In cases where cross-matching is not possible in emergency, a general guideline is that '0' group
recipient can receive any group, whereas A and AB recipient can be supplied with 'A' group or 'AB' (if
available) and for B group recipient only 'B' group platelet is to be supplied.
The rate of platelet transfusion is dependent on the circulatory status of the patient and while
calculating the amount of platelet or dosage required for transfusion, it is advisable to draw a correlation
between platelet count and bleeding time because they maintains a linear fashion. A platelet count below
50,000/cmm the bleeding time increases progressively.
Calculation of platelet requirement :
N = (PI-PO) x body surface area/IO
N =Number of units required
Po: Initial count of platelet
PI: Desired platelet count, usually estimated an hour after transfusion of platelet.
The dosage thus calculated is expressed in terms of platelet units,
Consumption usually increases with fever and those suffering from consumptive coagulopathy and
thrombocytopenia due to haemorrhage. In an average 4 to 6 random donor platelet units are required to
effectively reduce the bleeding. Pooled platelet manufactured by pooling of different group is not preferred .

[[p.21]]
342-
Blood Banking and Transfusion
because it is thought that Ag-Ab complex formed may induce clumping and it is advisable to use platelet
pooled within the same group. The single donor platelet of course is definitely the best.
Platelet transfusion (Table 11.4) can be autologus or homologus and their indications are described
later.
The platelets play a major role in haemostasis by adhering to subendothelial surface following
injuryto blood vesselsand helps in plugformationthroughadhesiveness(ref.Fig. on plateletaggregation)*
aggregation and promotion of intrinsic coagulation factor. But the mechanism is poorly understood and
needs further exploration.
The platelets are indicated in varieties of condition and again it varies with type of platelet transfused.
General indications are :
(a) Decreasedplateletproduction. :Leukaemia,aplasticanemia,radiochemotherapy,haematological.
(b) Thrombocytopenia: Haemorrhagic or dilutional,
(c) Idiopathic thrombocytopenic purpura.
(d) Qualitative defects :
(i) Inherited platelet type von willebrands disease, surface membrane defects (Glanzman's
thrombosthenia), granuledefects particularly a granule deficiency (grey platelet syndrome)
and many other conditions where platelet transfusion acts as supportive therapy.
(iii) Acquired: Myeloproliferative disorder, cardio pulmonary bypass surgery uremia.
Plasmapheresis is the. procedure by which plasma is selectively removed from anticoagulated whole
human blood and can be done by cell separators, centrifugation, filtration. The procedures employed to
separate plasma irrespective of technology yields about 200 to 250 rnI of plasma per unit of whole
blood.
Table 11.4. Platelet transfusion
Platelet concentrate preqfed from 450 In} of whole
blood consists 5.5 x 10
platelets per unit
I
I
Autologus platelet
Homologus
I
I
I
I
I
I I
I
(a) Solid tumours
Pooled
Washed
Monocyte
Irradiated
Frozen
Single
without involvement concentrate
platelet
poor
platelet
platelet
platelet
of bone marrow
(vol. 30-50 ml)
I
preparation
I
(b)' Haematological
I
I
malignancy
1. Commonly in
Suspected
I
leukaemia in
children
antibody
Immunocompromensed
remission
2. Those who
against
patient
I
can't bear with
PPF
HLA
Immunised
Commonly
circulation
matched
recipient
used now-
overload
cases
a-days to
prevent
TID and
alloim-
munisation
* Platelet plug inside an opened blood vessel. Primary platelet plug Fig. 2.6.

[[p.22]]
Blood Component Separation and Use
343
The donor criteria for plasmapheresis is same as that of selection criteria for the blood donors. The
mandatory tests are done prior to donation.
Plasmapheresis by centrifugation method is a simple procedure wherein the blood (350 to 450 ml)
is collected in ACD/CPD in a multiple plastic bag preferably double or tripple bag. The blood in the bag
is centrifuged while keeping the collection line ope~
with a saline drib once the centrifugation is over
the PRP (supernatant is primary bag) is transferred and retained in the first satellite bag. The RBC
concentrate is returned to the donor after separating the primary bag. The yield of plasmaIPRP in first
satellite bag is about ~OO to 250 ml, the yield can be increased upto 50 ml by repeating the procedure
again, double plasmapheresis.
Disadvantages
(i) Longer operative period.
(ii) Possibility of contamination is more.
(iii) Failure to return the red cells due to mechanical reason or inadvertant loss of RBC during the
procedure.
(iv) Chance of alloimmunisation and transfusion reaction is more, if collection is made from more
than one donor.
(v) Clerical mistake.
Advantage
Economically cheaper and simple procedure.
Plasma pheresls by filtration procedure
Membrane filtration devices using either flat bed filter or hollow fibres are in use for separating the
plasma from cell components. The blood (anticoagulated) is allowed to pass continuously at a high flow
rate through filter membrane. Plasma is separated and cells are filtered which are resuspended and
reinfused back. But the membrane filtration has the disadvantage of altering the cellular structure and
shorten theirin vivo survival after reinfused, so for component separation purpose, it is not recommended.
Disadvantages
Apart from the one mentioned above others are chance of contamination with deformed' platelet, cells
may leak out in case of damage to membrane. Also activation of complement may occur.
Advantages
Less clerical mistake as donors are not disconnected during operation. The procedure is technically
superior to manual plasma pheresis.
Plasmapheresis using cell separator
Cell separator machines using the centrifugation and filtration combinedly for plasma pheresis has been
developed and they can collect about 600 rnl of plasma in approximately thirty minutes time. They
operate in two phases using single venous access. Firstphase is collection phase and next one is reinfusion
phase and are ~utomatically
operated through computer control system. In first phase the anti-coagulated

[[p.23]]
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Blood Banking and Transfusion
whole human blood is pumped into separation device that uses a small membrane surface area for
separating the plasma which flows into collection chamber. In the second phase or reinfusion phase the
separated cellular component are collected into a reservoir upto the brim and further collection
discontinued, the blood pump reverses and reinfuses the cell components to same donor through same
venous access.
Advantages
Less operative period, donor friendly and user friendly closed automated procedure, almost nil
contamination in the separated plasma, no activation of complement and/or coagulation factor, less anti-
coagulant normal cell survival, less extracorpeal volume and particulate levels negligibly low.
Disadvantage
Very costly, requires a skilled operator and cannot be operated by anyone.
Uses and indications of plasma as component has been described earlier and will also be discussed
in appropriate chapter later on.
Neocyte processing by cell separator
Neocyte are the young, large and less dense RBC having a half life of 47.4 to 48 days with reticulocyte
count varying from 4.1 to 5%. It has been observed that these young red blood cells (1 to 10 days old)
has a 90 days life span whereas packed red cells in a unit has life span of 60 days.
These neocytes which are expressed earlier can be separated and processed by apheresis in celJ
separators operating continuously or intermittently.
Uses and indications
Neocytes are preferred in cases where repeated transfusion are the choice of treatment viz. thallasemia,
dyserythropoitic anemia etc., mainly to reduce the amount of iron deposition because by neocyte
transfusion frequency of blood transfusion can be reduced.
Therapeutic haemapheresis
This is the clinical application of haemapheresis employed selectively in treatment 'of certain specific
diseases either alone or in combination with other modalities of therapy, e.g., cytapheresis to remove
WBC in leukaemia, plateletapheresis in thrombocytosis, exchange of RBC in sickle. cell anemia and
plasma pheresis in so many conditions.
Therapeutic cytapheresis mainly applied clinically are :
(a) Leucapheresis
(b) Exchange of red cell
(c) Plateletapheresis.
Leucapheresis are mainly employed in haematological malignancy and in few other non-malignant
conditions as described below.
In chronic myeloid leukaemia the anti-coagulated blood is mixed with sedimenting agents and is
allowed to flow into separation chamber where myeloid leucocytes are effectively separated after which

[[p.24]]
Blood Component Separation and Use
345
the blood is reinfused back into the circulation. Usually repeated 2/3 times a week. This reduces the
hyperviscosity and leucostasis so common to CML and is observed in respiratory and neurological
system. Apheresis also has a favourable effect in reducing the hepato-splenomegaly and perepheral
count. However, this is used only as a short time supportive therapy to reduce the acuteness of pathological
process and thereby helping to achieve the benefit of chemotherapy and reduction of acuteness of
pathological process allows the patient to Jive a comfortable life. The curative therapy must continue
side by sid~.
Leucapheresis can similarly be employed in chronic lymphocytic leukaemia where addition of
sedimenting agents to input line of cell separator is not required. There is wider difference in specific
gravity between lymphocytes and erythrocytes, the separation of lymphocyte therefore occurs without
much difficulty. Mainly used in refractory cases to reduce the cell burden and hepatosplenomegaly, this
helps the patient to live a comfortable life but this is a short-term benefit only.
In acute myeloid leukaemia, where there is more than sufficient rise in count of WBe leucapheresis
helps these patients by reducing the cell burden approximately by 30 to 40% that helps to achieve better
result with chemotherapy. Survival and period of remission is thus improved.
Results of haemotherapy, therapeutic haemapheresis in hairy cell leukaemia is doubtful and needs
further evaluation.
Amongst the non-malignant condition the rheumatoid arthritis and sezary syndrome showed some
improvement in clinical manifestation with therapeutic haemapheresis but exact mechanism is poorly
understood.
Red cell exchange
This is frequently employed in cases of sickle cell anemia without any established proven value except
in cases of priapism and prior surgery. Partial exchange transfusion at an interval of 6-8 weeks when 500
ml blood was withdrawn and 500 ml red cells were reinfused were able to reduce the incidence of crisis
'(Anderson et al., quoted by Molison, seventh edition) and similarly partial exchange transfusion were
able to reduce eNS infarction. By partial exchange transfusion the main aim to achieve alevel of 60~
..
HbA was served. Ten of 15 children treated by Lusher et al., 1976 (quoted by Mollison) for a period of
,Ito 5 years showed improvement viz. remained free of pain crisis etc.
Plateletapheresis
This is mainly used in cases with thrombocytosis (increase in platelet count) and in chronic
myeloproliferative disorder. Thrombocytosis is usually observed in patient with splenomegaly and in
chronic myeloproliferative disorder with pregangrenous changes of extremities presumed to be due to
microvascular blockage or ischemic changes occurring immediately following blockage due to thrombotic
episode. In,these cases plateletapheresis is employed to reduce the platelet count.
Plasmapheresis
Therapeutic plasmapheresis or plasma exchange seemingly innocuous is rather a difficult procedure
than cytapheresis as it is frequently associated with complications like hypotension, urticaria,hypocalcimia
and infrequently with fatalities. By this procedure we remove harmful elements from plasma either in
free form or in bound form and unlike cytapheresis, it has proven value in treatment of certain pathological

[[p.25]]
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Blood Banking and Transfusion
processes viz. removal of antibodies, alloantibody, autoantibody, removal of monoclonal protein and
excess plasma constituent replacement of specific plasma constituent and miscellaneous condition.
Alloantibody commonly removed by plasmapheresis are anti-PLAI in cases of post-transfusion
purpura occurs usually within a week after transfusion. A fair trial for removal of anti-Rh in pregnant
women is still in experimental stage and its value vis-a-vis conventional procedure is yet to beestablished,
so not in common practice. So far autoantibody is concerned, it has a wider application in myasthenia
gravis where it can lower the level of autoantibody to acetylcholine, in goodpasteurs syndrome, it lowers
the autoantibody to glomerular basement membrane, autoantibody to perepheral nerve myelin that occurs
in guillain barre syndrome could be lowered by plasmapheresis and some of clinical manifestation were
relieved. Factor VIII inhibitors that causes uncontrolled bleeding even after infusion of factor VIII in
haemophiliacs and autoantibody in cold AIHA could be 'successfully removed by plasmapheresis.
Plasmapheresis has beneficial effect on waldenstonns macroglobulinimia (hyper viscous condition
due to monoclonal protein). At least two to three exchange of plasma is recommended.
On trial basis, it is being experimented in familial hypercholesterolaemia to remove LDL and is
found to have more beneficial effect with homozygous type of familial hypercholesterolaemia. Refsums
disease, a hereditary condition with inborn error of metabolism where phytanic acid is found in excess
responds well to plasmapheresis.
In thrombotic thrombocytopenic purpura characterised by fever, jaundice, purpura with
haematological picture of thrombocytopenia, hemolytic anemia and fragmented red cell mayor may not
be associated with renal pathology thought to be due to deficiency of prostacyclin or circulating antibody
to vascular endothelium, was given a fair trial with plasma pheresis and improvement, though observed,
is yet not established as modality for treatment.
Some conditions such a<; rheumatoid arthritis showed improvement by membrane plasma/pheresis
with cryofiltration. Mechanism of actions thought to be due to :
(i) Polymerisation of IgM antibody (cold temperature causes polymerisation), and
(ii) Lipids are filtered along with immunocomplex from the plasma. The filtered plasma is reinfused.
Other conditions like ITP and multiple sclerosis is undergoing trial.
Plasmapheresis thus plays a positive and definitive role in hyperviscosity syndrome, myasthenia
gravia and good pasteurs syndrome. In hyperlipidimia, it improves the condition. In rheumatoid arthritis,
SLE, Rh isoimrnunisation, AlHA and thrombotic thrombocytopenic purpura, polyneuropathy role is of
doubtful nature and needs to be ascertained properly and, clearly.
11.3 PLASMA AND PLASMA SUBSTITUTE
Plasma is the liquid component of whole human blood and makes up 55 to 60 per cent of total amount of
blood. The estimated plasma volume for man is 25 to 43 mllkg for women is 28 to 45 mllkg. This plasma
volume is variable in different conditions both physiological and pathological. Amongst the physiological
conditions, the plasma volume is higher in pregnancy and lower in stress dehydration and prolonged bed
rest. Pathological conditions are varied where the plasma volume shows definite fluctuation, for example,
it is towards higher side in congestive cardiac failure, nephritis, cirrhosis, in some cases ofpolycythaemia,
in some cases of splenomegaly, in cases of anemia, it may be high, normal or low, low in shock or in
cases of haemorrhage and perepheral circulatory failure. What has been described are very few amongst
a wide spectrum of conditions those are beyond perview of our discussion. Since the volume of plasma

[[p.26]]
Blood Component Separation and Use
347
and/or its different fraction varies with the prevailing pathological process, a need for its replenishment
may arise in connection with therapeutic necessity. And all the time fresh plasma may not be available
readily. To tide over such emergent situation, alternative was conceived. The outcome was plasma
substitute designed to provide colloid osmotic pressure or expand the plasma volume.
Freeze dried plasma and/or lyophilised pooled plasma, plasma protein solutions, concentrated
fractions like albumin, factor VIII etc.
Plasma substitute
They can be colloids or crystalloids which can act as plasma substitute and are available commercially.
Colloids provide colloidal osmotic pressure and products available in market are Dextran 70, Dextran 5,
HES or hydroxyethyl starch and gelatin.
Dextrans are liquid with high molecular weight, high oncotic pressure and required viscosity but
they can tide over the emergency as a plasma substitute only and not as effective as human plasma. They
are available as LMW Dextran - Dextran 40 and HMW Dextran - Dextran 70 with half life of 6 to 8
hours respectively.
The LMW is available in bottles, plastic container asceptically packed and sealed, dispensed as
10% in isotonic saline or in 5% dextrose in water for injection to be used parenterally through intravenous
route. To start with administration may be done rapidly in the first hour and maintained at 12 to 14 mllkg
per day preferably limited to 500 mVday in an adult human. It has been observed that itdecreases the
viscosity of blood, improves circulation in capiJIary and microvasculatures, may play some role in
preventing thrombosis. It is rapidly excreted in urine.
The HMW is also available in bottles/plastic container packed and sealed asceptically, dispensed as
6% solution in isotonic saline, it has prolonged action because it is excreted comparatively, slowly in
urine. It interferes with blood coagulation mechanisms and has antithrombotic action. Total administration
is to be limited within 1500 ml of Dextran 70 and instead of continuous administration, it should be
interrupted with crystalloid administration. 500 mlIday is a preferable dose unless indicated otherwise.
At this dosage, it offers a prolonged action and protection against thromboembolic episode.
.
Advantages are many viz., ready availability, contamination free as such very less chance of TID,
need not worry about compatibility of transfusate.
Disadvantages are circulatory overload, alergic reaction. In dehydrated patient, it may accentuate
tubular obstruction, coagulation defect due to inhibitory effect on plaetelet function, causes interstitial
dehydration, prolongation of bleeding time.
Contraindication
Cases with renal pathology, congestive cardiac failure, dehydration, known cases of thrombocytopenia.
Preceution
Since Dextran may cause interstitial dehydration a continuous administration is not advisable but if
condition warrants, it may be combined with crystalloid administration in the intervening period.
HES (hydroxyethyl starch)
Recently introduced HES 450, available as6% solution in isotonic saline. It is a moderate expander with
a longer half life, M.W. 45000.

[[p.27]]
348
Blood Banking and Transfusion
Disadvantage
Severe allergy, coaguJation defect, no antithrornbotic property.
Advantage
Renal function remains unimpaired. Dose should not exceed 3500 ml/day,
Gelatin
Available (in plastic container) as haemaccel in 500 ml pack as 3.5 to 4% solution. A moderate expander
with MW 35000 and half life 4 to 5 hours.
.
Advantage
No haernostatic defect observed, non-antigenic, renal function unimpaired, no antithrombotic effect
seen.
Disadvantage
Circulatory overload, infrequent allergic reaction.
Dosage recommended is upto 1000 ml/day,
Crystalloid
Plasma consists of 90 to 92% water and 8 to 10% organic and inorganic substances. Major part of
organic substance are protein, albumin, globulin and fibrinogen apart from glucose, fat like substances,
aminoacids, metabolites, enzyme, hormone. Inorganic substance approximately 0.9 to 1% and is comprised
of salts of sodium, potassium, calcium. Sodium chloride play the main role in keeping osmotic pressure
of blood relatively constant. In normal human being osmotic pressure is maintained at a constant level in
the tissues and any change in osmotic pressure will interfere with normal activity.
Crystalloid solutions that are commonly used as volume expander and makes up for the deficit
temporarily are 0.9% sodium chloride and lactated Ringer's solution. Total body fluid is broadly divided
into intracellular fluid and extracellular fluid. The later is further subdivided in intravascular (plasma)
and interstitial (fluid in tissues between blood vessels and cells). In plasma and interstitial fluid main
cation is sodium and main anion is chloride. Normally cellular metabolic process maintains the ionic
composition of intracellular fluid and extracellular fluid. Any disturbance will, therefore, induce
disharmony in maintenance of osmotic equilibrium. Large quantity of fluid is required to expand the
plasma volume. The crystalloid expand plasma volume temporarily so a tendency to administer more of
crystalloid is to be avoided.because this may cause fluid overload and flood extravascular space resulting
in oedema.
0.9% sodium chloride is administered LV. @ 400 mllhour in adult total, dose is to be adjusted
depending on condition. Lactated Ringer's given LV. @ 5 to 10 ml per kg per hour and total dose limited
to 1to 3 litres per day in adult.
Normal human plasma and commercial plasma
Normal human plasma is an opalescent liquid, colourless or mildly yellowish 'should not contain any
visible particle except the precipitated fibrin that may be observed after long standing. It is the fluid part
of whole human blood that has been stabilised against clotting (anti-coagulated) and separated from
solid/cellular element of whole. human blood.

[[p.28]]
Blood Component Separation and Use
349
It is available as single donor plasma or as pooled plasma lyophiJised. The former·is conventionally
prepared by centrifugation and subsequent separation whereas pooled plasma can be prepared by cell
separator in smaller scale but for commercial use, it is prepared in a larger scale in plasma manufacturing
unit.
Pooled plasma is prepared by pooling equal amount of anticoagulated plasma from whole human
blood. These plasma pooled should not necessarily be fresh and commonly pooled from recovered unit,
..unutilised unit tested and cleared for all mandatory test. But there are certain guidelines in this regard
which are to be· adhered to while separating plasma.
(a) Blood should not show haemolysis or bacterial contamination.
(b) Storage temperature for source blood unit must not exceed 10°C. Preferably stored at I ° to 6°C
for 4 weeks.
(c) Cell free plasma to be stored at a temperature range of 1°C to 6°C.
(d) Pooling should be so done that plasma from groups A, 0 and either B or AB should be in a
proportion of 9 : 9.2 (A : 0 : BlAB) and pooling is done in a strictly asceptic condition.
(e) Pooled plasma dispensed in suitable containers sealed and packed asceptically should contain
4.5% w/v of protein, haemoglobin 0.025% w/v, pyrogen free and sterile and comply with
identification test.
Labellings essential and so before the product is checked out, it consists of following description
the anticoagulant used, number of donors, protein content, storage condition. Checked for sterility and
pyrogen. Manufacturing and expiry date, Licence number..
Lyophilised pooled plasma has been discussed in section 10.1. The lyophilised pooled plasma is
categorised as plasma volume restorer. It is available as pale cream powder or as a friable egglomerate.
It has to be reconstituted before use. The volume of plasma before lyophilisation, if 250 mI, then the
water for injection for reconstitution is recommended 250 ml. It is better to follow instructionon the
label. The diluent water is added slowly by the side of container and 'diluent should preferably be at
20°C. Simultaneous gentle shaking of container is to be done to avoid froth formation. The procedure of
reconstitution is continued for ten minutes. A pale yellow coloured fluid is obtained which should be
used immediately without delay.
.
The fluid should comply with standard set for protein (4.5% w/v), haemoglobin (not more than
0.025% w/v), loss for drying (not more than 0.5%), that can be determined by drying 2.0 gms for 24
hours in vacuum), sterile and free of pyrogen and has to be identified.
Identification
Lyophilised pooled 'plasma reconstitute can be identified by testing with specific antisera precipitation,
tests should show that it consists only human plasma protein.
It can also be identified by addition 0.2 ml calcium chloride to 1 ml of reconstituted solution,
coagulation should occur which can be catalysed by heating at 37°C ~,yophilised
pooled plasma prior to
reconstitution is stored in an atmosphere ofnitrogen in a sterile container which is light resistant. Contents
of the container are sealed and packed in a cool place under strict asceptic condition so as to avoid
contamination with microorganism.

[[p.29]]
350
Blood Banking and Transfusion
Label states
(a) Anticoagulant status
(b) Volume of plasma after reconstitution
(c) Volume of water for injection to be added
(d) Total number of donors or the source from where plasma collected
(e) Use immediately after the reconstitution
(f) Date of manufacture and expiry
(g) Licence No.
(h) Storage condition
(i) Checked for sterility and pyrogen
G) Special instructions, if any, viz., transport condition that the contents are free of hepatitis and
HIV.
Advantage
Readily procurable, has all the biological property that a normal human plasma should have, need not go
for compatibility testing, ten minutes is required for reconstitution and fluid is ready for use, reconstitution
procedure is easy and is done with water for injection only; easy transport and storage, extended shelf
life.
Disadvantages
Chances of hepatitis and mv cannot be ruled out easily in any pooled product even though it is certified.
Plasma ·protein 'solutlon(PPS)
They can be prepared from pooled plasma after removal of factor VIII, fibrinogen and immunoglobulin
fraction. The plasma fractionation cannot be done in a.blood bank because a large amount of pooled
plasma that vary according to demand viz. 50 litres to 1000 litres. To process this amount of plasma,
proper industrial set up with necessary requisites such as temperature, controlled environment, manpower
and clean- sterile set up is essential. The infrastructure for quality assurance is also required. The two
methods of fractionation are in use :
(i) Cohn's ethanol fractionation method started by Dr. E.l. Cohn in 1940 and perfected by Cohn et
ale in 1945.
(ii) Chromatographic method.
Operational flow of both the procedure is beyond the scope of this section.
Majorcontent ofPPS is albumin and are available commercially as described below. PPSare prepared
from liquid pooled plasma that has been obtained from healthy donors who are screened properly for
collection of whole human blood and the whole human blood, thus collected and used as a source has to
be cleared for all mandatory test. This liquid plasma need not necessarily be within expiry period and
may be unutilised plasma, recovered plasma, the plasma that have just crossed the expiry date but within
40 days of date of collection, stored at a temperature of 2°C to 6°C.
Commercial products available in sterile glass container, dispensed mostly in 100 ml bottles. The
contents of the bottle are packed and sealed under asceptic condition.
(i) 5% albumin
(ii) 25% albumin

[[p.30]]
Blood component Separation and Use
351
These are albumin concentration and has to be diluted with 5% electrolyte solution before being
infused through I.V. route. The solution is compatible with whole blood, packed RBC and carbohydrate
for injection but non-compatible with alcohol and protein hydrolysate. Some preparation of salt poor
albumin are also available. In 25% albumin, the major constituent is 96%. Albumin and rest is 4%
globulin.
Most important property ofalbumin in vivo is that it is responsible for 80%plasma colloidal osmotic
pressure and thus "thealbumin is categorised asblood volume supporter.
Albuniin available commercially is administered IV after reconstitution. Reconstituted solution must
be used within 4 hours of reconstitution.
Dosage varies according to the disease and condition of the patient. The general guideline for dosage
of 5% albumin is that it is given in two phases :
(a) Initial dose
(b)' Maintenance dose.
The initial dose in adult is 200 to 500 ml which is again dependent on the condition of the patient.
Maintenance dose in adult 0.4 gm to 0.6 gm per kg of body weight administered I.V. @ 50 to 100 drops
per minute of reconstituted solution. The initial dose in children is comparatively less and preferably
"limited to a total dose of 25 to 35 ml, 0.5 gm to 0.6 gmlkg of body weight is adrriinistered I.V. as initial
dose. The maintenance dose in children is lower and is decided by paedeatrician on the merit of each
case.
Plasma proteinfraction (PPF) is a blood volume supporter transparent, colourless, odourless liquid.
May develop granular flaky deposit, if stored for long period.
It is a sterile solution available as 5% solution in electrolyte, major constituent is albumin, 83% and
rest 17% is globulin (exglobulin and ~ globulin), it exerts colloid osmotic pressure similar to pooled
plasma.' It contains total protein ranging from 4.3% to 5.3% w/v of total protein (83% albumin, 17%
globulin - alpha globulin, beta globulin and gamma globulin should not exceed 1%); potassium ion may
be present but not 'more than 2 millimole per litre. No fibrinogen and antibody are present. Donor
criteria same as above. It may be prepared by any of the"two methods.named above viz., Cohn's method,
liquid chromatography. Final product has pa·between 6.7 to 7.3, before dispensing, it is sterilised by
filtration and dispensed in glass containers under asceptic condition and environment. Sealed containers
are now heat treated for ten hours continuously at a temperature ranging from 59.SoC to 60.5°C to
inactivatemv and hepatitis virus. After that, containers are again incubated at 30°C to 32°C for 14 days
and observed for any physical change - colour, turbidity, presence of foreign particle etc. If change is
observed, products are declared unfit for human consumption and discarded.
Sterilisation, heat treatment and incubation are common for all products and has to be strictly observed.
Storage shelflife can be extended by storing at lower..temperature. It is to be stored away from light.
At a temperature of 2°C to 8~.c
all the products can be stored for five years but at a temperature 20°C to
25°C, they can be stored.only for three years.
-Labelling
The label should contain following particulars :
(a) The volume
(b) The total protein

[[p.31]]
352
Blood Banking and Transfusion
(c) Sodium concentration and potassium concentration
(d) .Stabilising agent, if used, has t<? be mentioned
(e) Source material
(f) Storage particulars
(g) Manufacture and expiry date
(h) Conditions or instructions for use LV. administration, not to be used if turbid, to be used within
4 hours after opening of the seal
(i) 'Licence no. for manufacture.
Use and indications
For 5% albumin and PPF common indications are as follows:
1. Shock: To restore volume loss, when blood and other plasma substitute not readily available.
5% albumin solution may be administered LV. to combat the circulatory failure.
2. Bunts: During first 24 hours more crystalloid and less albumin is commonly recommended.
After 24 hours, it is just reverse more protein (albumin) and less crystalloid.
3. The replacement fluid in plasma exchange.
4. Acute nephrosis: Albumin and diuretics are used as supportive therapy.
5. HDN: In HDN the use of albumin though not well established but are administered to help
detoxification by binding bilirubin.
6. By pass operation : In cardiopulmonary bypass the priming of pump is sometimes done with
albumin 'and crystalloid to adjust the haematocrit and albumin level in plasma of the patient.
7. Respiratory distress syndrome: Albumin and diuretic 'assupportive therapy in the hypoprotinimic
fluid overload.
25% albumin special indication and ~sage
(a) Severe hypoprotinimia : Nephrotic syndrome (described above) andhepatic disorder.
(b) Severe hyperbilirubinimia in newborn (described above) : Salt poor albumin is recommended.
(c) Pre-eclampsia: Salt poor albumin is recommended when clearly indicated.
Contraindication
The hypoprotinimia, when staoiused as observed in case ofcirrhosis of liver, chronic nephrotic syndrome:
malnutrition due to protein loosing enteropathy or malabsorption. In cases of congestive cardiac failure
associated with severe anemia.
'
Side effect
Administration of albumin, PPF etc. may be associated with hazards like allergic reaction, fluid overload,
patient may sometime become febrile orthere may be sudden and abrupt change in pressure thought to
be caused by release of vasoactive substances (common with PPF) so while administering through I.V.
route take due precaution not to infuse very rapidly a large amount.

[[p.32]]
Blood Component Separation and Use
353
11.4 BLOOD SUBSTITUTE
The whole human blood is collected from donors, stored for a limited period and is transfused within
that period either as whole blood or as component. The limited supply of blood cannot all the time meet
with the ever increasing demand due to injury (accidental or otherwise), surgery and many other medical
condition where blood or its derivatives are being used increasingly as a therapy or supported to cure or
palliate many a conditions. Again the blood or its components are to be processed through mandatory
checking and cross matched before transfusion. Transfusion of blood of human origin is frequently
associated with hazard. So to overcome the short supply, eliminate crossmatching and the transmission
of disease through transfusion, the scientist and researchers are in search for a blood substitute which
can fulfill the above criteria and at the same time, can perform the most important function (oxygen
carriage) of blood and serve the emergency needs at least for a short while till the compatible tested
blood is made available to the patient.
After much research, it was observed that for emergency purpose, the two functions of blood has to
be attended immediately and they are respectively maintenance of blood volume and oxygen carriage.
So the various blood substitute which are either of human origin or artificially produced are divided into
volume expanders and oxygen carriers from functional point of view. The VOIUi11e expanders has already
been discussed in previous section and therefore, in this section, we briefly discuss about the oxygen
carriers.
As shown in Table 11.5, we can get oxygen carrying blood substitute from two sources:
(a) Biosource : Haemoglobin is the compound that carries oxygen.
(b) Artificial source: Chemical substance of inert nature that have the capacity to carry oxygen.
Biosource
In human blood haemoglobin is the compound that carries oxygen. Thus, a blood substitute derived
from any biosource must have haemoglobin as an essential component to serve the function of oxygen
carrying. These biosubstitutes of blood could be processed from:
(a) Sources other than human.
(b) Human sources.
In sources other than human the animal source and bacterial source are exploited for production of
haemoglobin on experimental basis and is still under trial. A brief discussion is given below.
In animals through genetic engineering, the genetic materials are inserted into animals inducing
them to produce animal red blood cells containing human haemoglobin.
In the other one a recombinant human haemodilution factor (rHb-I) is produced in E. coli culture.
Through molecular engineering, glycine is inserted between two alpha chain ends and thereby disallowing
the breakage of haemoglobin into toxic dimers in the kidney. And substitution of lysine at 108 position
by asparagine enhances the oxygen carrying property of the produce more than RBC.
Both the above products are in experimental stage and if perfected will have the advantage of :
(i) Less human donor
(ii) Less chances of TID
(iii) Immediate use of the product in emergency.

[[p.33]]
354
Blood Banking and Transfusion
The haemoglobin derived from human source are unmodified haemoglobin, modified haemoglobin
and encapsulated haemoglobin.
While experimenting, it was observed that stroma with haemoglobin is the stumbling block in
processing the blood substitute. The stroma frequently causes. hypercoagulopathy and as such stroma
free haemoglobin (SFH) was prepared. But the free haemoglobin in SFH has short life span of less than
three hours in vivo and can be thus used on a short term basis to tide over the emergency situation till
blood is made available for the patient. The technique of preparing SFH has been perfected by institute
of immunohaematology (ICMR, Mumbai) in India.
Advantages
(i) Renal shutdown by toxic dimers of haemoglobin (breakdown of haemoglobin produces toxic
dimers in blood substitute) has not been observed with SFH.
(ii) Can meet emergency requirement of whole blood.
(iii) Group specificity is not needed.
Disadvantages
(i) Haemoglobin in SFH solution shows higher affinity for oxygen and oxygen release to tissue is
thus lessened.
(ii) Quick clearance from plasma.
Modified haemoglobin and encapsulated haemoglobin are still in experimental stage and under
trial.
In modified haemoglobin, haemoglobin has been modified by reducing its oxygen affinity through
pyridoxylation and increasing the size of haemoglobin to delay the clearance. The increase in size is
done either by polymerisation ofhaemoglobin molecule or byconjugationofhaemoglobin with polymers
preferably biocompatible.
'
In encapsulated haemoglobin, the haemoglobin is encapsulated within liposome membrane. and it
gave good result in experimental animal. For human use an improved quality refined membrane is
required.
The modified haemoglobin and encapsulated haemoglobin is in experimental stage and is yet to be
used in human being.
Artificial sources
The artificialsource could be chemical compound only and since it is for human consumption, it must
be of inert nature with property of good affinity and avidity for oxygen that could be transfused at ease
in room temperature or at best in body temperature. It must be compatible with whole human blood.
After long research, it was observed that the perfluoro compounds meets some of the criteria as
described above. The compound is a substance with inert nature. Two of perfluorochemical has been
choosen:
(i) Perfluorotrybutylamine and the other one is
(ii) Perfluorodecalin.

[[p.34]]
Blood Component Separation and Use
355
The former remains in tissue for long time and the latter rapidly cleared through lung. The main
disadvantage with perfluorochemicals is that they require high oxygen tension for effective carriage of
oxygen; this is usually fraught with less oxygen release and maintenance of patient in a high oxygen
tension, potentially toxic. Apart from this, emulsions are unstable at room temperature so storing in
frozen state is essential. Further the perfluorochemicals are not soluble in water.
Toovercome all this and bring it nearer to the requirement a compound Fluosol DA has been developed
in Japan. In this compound, both the perfluorochemicals described, i.e., perfluorotrybutylamine and
perfluorodecalin are used at a concentration of 6% and 14% respectively with emulsifying agents like
pluronic F-68 and yolk phospholipids, oncotic agents like hydroxyethyl starch and for electrolyte the
Ringer's solution are also added. This is undergoing extensive trial but has not yet been cleared for
. general use as blood substitute. They are mostl~
used for research purpose.
The common blood substitute in general use on a short term basis is stroma free haemoglobin. This
fulfills the emergency requirement of oxygen carriage but are unable to replace substitute the whole
human blood for long term use. The shelf life is not limited and can be extended as long as the solution
can be kept sterile, lyophilised form can be stored at room temperature. And since the group specificity
is not needed, it can be made readily available in field to tide over the emergency situation.
Books recommended for further reading
1. AABB: Technical Manual, 10th edition, Washington DC, 1990.
2. BloodTransfusion in Clinical Medicine, P.L Mollison, seventh edition, 1983,Blackwell Scientific Publication,
UK.
3. Blood Group Serology, Boorman, Dodd and lincoln, sixth edition, 1988, Churchill Livingstone.
4. Clinical Diagnosis and Management by Laboratory Methods ofTodd; SanfordandDavidshon; John Bernard,
second Indian edition, 1989, ArrBS.
5. Development Strategies on Blood Transfusion Services in India, R.N. Makrool, sponsored by WHO, 1988,
Parallel Time Editorial Agency.
6. Introduction to Transfusion Medicine, Bharucha and Chauhan, first edition, 1990, D.K. Publishers, Mumbai-
400033.
7. Transfusion Medicine Technical Manual, Saran andMakroo, 1991, sponsored by WHO, Publishedby DGHS,
Nirman Bhawan, New Delhi.
Journals and articles
1. Bharucha, Zarin, S., Application of Cell Separator Machine Immunohaematology, Bulletin, Vol. 14, No.6,
pp. 1-4, Nov.-Dec., 1983.
2. Bharucha, Zarin S., Recent Development in Transfusion Medicine lmmunohaematology, Bulletin, Vol. 20,
No.1, pp. 2-8, Jan.-Feb. 1989.
.
3. Dutra, R.N. (Brigadier), Platelet Transfusion in Thrombocytopenic Haemorrhagic Disorder,
Immunohaematology Bulletin, Vol.1, 7, No. J, pp. 1 to 5, Jan.-Feb. 1986.
4. Transfusion Bulletin, Vol. 5, No.4, p. 14-16, Jan.-Mar., 1993.
5. Transfusion Bulletin, Vol. 6, No.1, p. 2-5, April-June, 1993.

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Blood Banking and Transfusion
Advertisements and information booklet
1. Efficient Utilisation ofBlood, Courtesy: BharatSerum and Vaccines, 27Road;Wagle Estate, Thane, Mumbai.
2. Product Information Booklet, HajJkein Biopharmaceutical Corporation Limited, Parle, Mumbai, pp. 20,
effectiv from June 1991.
3. Haemonitics \t'50 Apharesis System, Consolidated Product Corpo ration, Chiranjib Tower, Nehru Place,
New Delhi.
4. Auto Pharesis, C. (Plasma Pharesis System), Fenwal Laboratories Division of Travenol Laboratories Inc.,
Illinois, USA.
5. CS 3000 Blood Cell Separator, Fenwal Laboratories, Division of Travenol Laboratories Inc., Illinois, USA.