Anticoagulants

• ANTICOAGULANT: Chemical that prevent
clotting of blood
• PRESERVATIVE: a chemical added to any
specimen to prevent changes in the
constituents of it
• In the body, blood is in fluid state due to
optimal temperature, pH, proper nourishment
to cells and detoxification.

• Before the discovery of anticoagulants, defibrinated
blood was used(whipping & twirling of collected blood)
• Direct transfusions happened which needed great
surgical expertise
• Birchoff (1835) did transfusion of defibrinated blood in
animals
• In JOHN HOPKINS HOSPITAL: collected blood was
agitated in Erlenmeyer flask with glass beads till clot is
fully formed & clot was removed; remaining liquid
portion with RBCs poured into another container
through a sterile gauze & and used for transfusion
• Till 1920’s defibrinated blood was used for transfusion

• Transfusion reactions occurred because of
damage of RBCs due to trauma to collected
blood during defibrination
• CURTIS & DAVIS(1911): used Y-shaped paraffin
coated cannula tied into veins of both donor &
recipient & a syringe was attached to neck of
cannula
• KIMPTON BROWN(1913): used paraffin coated
glass cylinders with horizontal side tube for
suction
• BERNHIEM: Syringe , U Shaped Tubes for needle
attachment & 2 way Stopcock Apparatus
• UNGER: Syringe, 4 way outlet Stopcock(blood
aspiration, injection to recipient & for flush out)
Curtis & Davis
Bernheim

• JOHN BRAXTON HICKS(LONDON,1868): First to
experiment with chemical methods to prevent
coagulation of blood ; phosphate of sodas(blood kept
fluid but patient died of shock)
• Swiss Physiologists ARTHUR & PAGES(1890):
Connect calcium with blood clotting on addition of
any small amount of organic salts
• LANDOIS(1892): Suggested Hirudin from leeches
• A.E.WRIGHT(1894): Suggested non-toxic citrates;
that bind enough ca without causing convulsions [21
years back before it was used]
• SATTERLEE & HOOKER(1914): Used Hirudin but
found to have a narrow therapeutic window&
difficulty in obtaining

• WEIL(NY,1914): Citrated blood can be
preserved in icebox for long term basis
• HUSTIN(BELGIUM,1914): Reported use of
sodium citrate & glucose
• AGOTE(ARGENTINA,1915): Citrate added
blood can be kept for longer time
• LEWISOHN(NY,1915): Minimum amount of
0.2% citrate can prevent clotting & relatively
safer in massive transfusions with 2.5litre of
citrated blood
ALBERT HUSTIN
LUIS AGOTE
RICHARD WEIL
RICHARD
LEWISOHN
LEWISOHN & ROSENTHAL(1933):
Explained that hyperpyrexial reactions
occuring is because of bacterial growth
in distilled water which was being used
as a solvent for anticoagulant

YEAR PERSON ANTICOAGULANT REMARKS
1869
Braxton Hicks Sodium
Phosphate
1st example of blood
preservation research
1914 Hustin Sodium Citrate Citrate used 1st time
1915 Lewisohn Sodium Citrate Determined
minimum amount
needed for
anticoagulation and
demonstrated non
toxicity in small
amounts
1916 Rous and
Turner
Citrate -dextrose 1st anticoagulant
1917 Robertson Citrate-Dextrose Used RT solution to
build blood bank in
Harvard medical unit
1943 Loutit and
Mollison
(ACD)Acid citrate
dextrose
Lowered the pH to
5 to make ACD
1950 Carl Walter & W P
Murphy
Plastic blood bags Easy
transportation &
component
preparation
possible
1950 Audrey Smith Cryopreservation
of red cells using
glycerol
Long term storage
1957 Gibson Citrate phosphate
dextrose
Cells passively lose
phosphate , so by
adding phosphate
this loss can be
prevented
1957 Gabrio and
collegues
Inclusion of
nucleotides
Important for red
cell metabolism
1968 Shields CPDA-1 Showed that
Adenine
(preservative)
markedly improved
WB storage
1970 Beutler Concept of additive
system
1979 Hogman (SAG)Saline , adenine,
glucose
5 weeks storage
without adverse
effects of high ph
1980 Lovric CP2D in primary bag with
additive solution
composed of SAG , sodium
citrate , citric acid and
sodium phosphate
1985 ---- SAGM Shelf life reduced
to42 days

AUDREY SMITHPEYTON ROUS PATRICK MOLLISON
JOHN G GIBSON

IN-VITRO STUDIES
• Stored RBC studied using biochemical and
morphologic measures
• Safer studies
• Lower expense
• Standard used: Hemolysis less than 1% in US
and 0.8% in Europe
IN VIVO STUDIES
•Measuring RBC recovery and survival in
volunteers (HUMAN TRIALS)
•Are of greater than minimal risk
•Require scientific, medical and radiation
physics oversight
•Clear standards:- 75% survival after24 hours
and hemolysis less than 1 %
•Disadvantage :- need for stable population of
volunteers

STORAGE LESIONS
ATP 2,3 BPG
•Constellation of changes associated with
irreversible erythrocyte damage and reduced post
transfusion survival following storage at 2 -6O c
They have effect on :
1. Post transfusion viability reflected in ATP levels
2. Hemoglobin function reflected by 2,3 DPG

PHYSICAL BIOCHEMICAL
Morphological changes-Disc
changes to echinocytes and
then to spherocytes
Decrease in 2,3 DPG-Decrease
in pH-Shift of O2 dissociation
curve to left-Decrease release
of O2 from Hb to tissues
Change in deformability due to
loss in membrane lipids
Electrolytes-poor functioning
of Na-K+ Pump-accumulation
of K+ in stored blood
Increase in osmotic fragility Oxidative damage with change
to the structure of Band 3 and
Lipid peroxidation
PHYSICAL
Loss of heat labile coagulation factors ie
factor V and VIII(30% and 20% respectively)
Apoptic changes with racemization of
membrane phospholipids

CONSEQUENCES OF
STORAGE LESIONS
1. These events risk compromising
the safety and efficacy of long
stored RBC
2. Reduces their capacity to carry
and release oxygen
3. Promotes release of potentially
toxic intermediates
4. Negatively influences on
physiological rheology

COMMON ANTICOAGULANTS USED
IN HEMATOLOGY
• EDTA
• SODIUM CITRATE
• HEPARIN
• OXALATES
• SODIUM FLUORIDE
• IODOACETATE

EDTA
• Its used in a concentration of 1 to 2 mg/ml of
blood
• It may be used for both hematology and
chemistry tests, and is the preferred
anticoagulant for blood cell counts (CBC), ABO
Blood grouping.
• Cytotoxic and weakly genotoxic

SODIUM CITRATE
• The ratio is one part of 3.8% aqueous solution
to nine parts of whole blood.
• Its used for blood coagulation and platelet
function studies (PT &PTT) and can be used
for Erythrocyte sedimentation rate (ESR).

OXALATES
• Inhibit blood coagulation by forming insoluble
complex with calcium
• K oxalate: concentration of 1-2 mg/ml of blood
• NH4+ & K+ oxalate mixture in the ratio of 3:2, and
2 mg / ml of blood is the required amount
• Ammonium and/or potassium oxalate does not
cause shrinkage of erythrocytes, consists of three
parts by weight of NH4+ oxalate, which causes
swelling of the erythrocytes, balanced by two
parts of K oxalate which causes shrinkage

• SODIUM FLUORIDE
 weak anticoagulant but is often added as preservative for
blood glucose together with potassium oxalate as
anticoagulant
 It is effective at a concentration of 2mg/ml blood
 It inhibits the enzyme enolase (sodium fluoride) thus
inhibiting glycolysis. Na fluoride/K oxalates are mixed in the
ratio 1:3
• IODOACETATE
 Sodium iodoacetate at concentration of 2mg/ml is an
effective antiglycolytic agent and a substitute for sodium
fluoride. It is a potent (suicide) inhibitor of G3P
dehydrogenase
 Formalin, toluene, 6N HCl, Boric acid, thymol, chloroform
are used as urine preservatives for 24 hour urine

Stopper color Additive Notes
Red No additive •Used for blood bank,
some chemistries.
•Collection of serum
•10-15 min is required to
allow blood to clot
before centrifugation
Lavender
(purple)
EDTA •Collection of whole
blood ( binds calcium)

Green Sodium or lithium
heparin
•Inhibits thrombin
activation.
•chemistry studies
Light blue Sodium citrate •Coagulation studies
(bind calcium) (PT &PTT)
(ESR).

Gray •Sodium fluoride &
potassium oxalate: inhibits
enolase (phosphopyrovate
dehydrogenase)
•Sodium iodoacetate:
inhibits glocose-3-
phosphate dehydrogenase
•For glucose
determination in
chemistry
(stabilize glucose in
plasma)
Yellow Acid citrate dextrose
(ACD)
•For use in blood
bank studies, HLA
phenotyping, DNA
and paternity
testing
(preserves red cells)

HEPARIN
• Prevents coagulation by inactivating the
prophylactic activity of thrombin after combining
with AT 111 and thrombin.
• 1000 IU of heparin is equal to 10 mg (commercially
available as IU)
• Dose of heparin for anticoagulation is 0.5-2.0 IU/ml
of blood (approx.500 IU of heparin for 500 ml of
blood)
• Heparinized blood should be used within 24 hours.
• Earlier heparinized blood was used in pen heart
surgery but now usually it is not used as
extracorporeal pumps are now usually primed with
crystalloids and not with blood.
•The effect of heparin can be neutralized with
Protamine sulphate
•1 mg of protamine sulphate neutralizes 1 mg of
heparin (to neutralize 5000 units of heparin [50 mg], 5
ml of 1 % solution of protamine sulphate will be
needed)
•Heparin is used in cord blood collection apart from
CPD

CITRATE DEXTROSE
• First anticoagulant preservative
• Introduced in 1916 (World War 1) by Rous and Turner
• Initially used to store rabbit blood
• Blood stored up to 12 days in citrate-sucrose solution
prevented anemia post transfusion.
• Dextrose was found to be marginally better in diminishing lysis
in human blood
ADVANTAGES :
• Most efficient and available anticoagulant for greater part of 20
years
DISADVANTAGES :
• Required dilution of blood to about three times its original
volume (1:3)
• Could not be heat sterilized because sugar got caramelized
• Risk of bacterial contamination from open mixing of ingredients
and addition of solution to the bottle

ACD (ACID-CITRATE-DEXTROSE)
• 1943(world war II) Loutit and Mollison showed that simple
acidification of citrate dextrose prevented caramelization of sugar
• pH of citrate dextrose was lowered to make it 5
• ACD first used in 1:4 ratio but later concentrated to use in 1:7
ratio
• ACD blood was the basis of building national blood systems in
British commonwealth and USA during World War II
• ADVANTAGES:
– Simplified sterilization procedure
– Reduced volume of preserving solution
– Enhanced preservative properties
– Increased shelf to 21 days
– Minimal effects on acid base balance
• DISADVANTAGES :
– Levels of 2,3 DPG lost early within 1st week

• ACD is used during procedures such
as plasmapheresis instead of heparin
• Two different solutions (Solution A and B) are
defined by the United States Pharmacopeia
CONTENTS (g) ACD-A ACD-B
Total Citrate 20.59 to 22.75 12.37 to 13.67
Dextrose 23.28 to 25.73 13.96 to 15.44
Sodium 4.90 to 5.42 2.94 to 3.25

CITRATE PHOSPHATE DEXTROSE
• John .G . Gibson developed a method for estimating in
vivo red cell survival using double isotope procedure
with 51 Cr and 32 P to study the effect of blood
preservative solution & noted existence of high energy
phosphate compound in blood collected in CPD (later
found to be 2,3 DPG)
• Because of the lower pH in the ACD preservative, most
of the 2,3-DPG is lost early in the first week of storage &
thus CPD came into widespread use in the US in 1970s
because it was superior for preserving 2,3 BPG since it
has a higher pH
• Even in CPD, RBCs become low in 2,3-DPG by the second
week.
• Shelf-life of blood stored in CPD at 2-4 °C was 28 days
with <30% RBC loss

• ADVANTAGES :
I. Better maintenance of 2,3DPG
II. Higher pH (5.6)
III. Improved ATP synthesis
• CPD with 16 mMol/L phosphate increased
the fraction of RBC recovered after 3 weeks
of storage from75% with ACD to more than
79%,and after 4 weeks only slightly lower
than storage in ACD

ADENINE (CPDA- 1)
• CPDA - 1 was developed in 1968 and shown
to permit whole blood storage for 5 weeks
• However regulatory questions regarding
safety of adenine (especially development of
uric acid stones) delayed licensure of CPDA 1
in USA for 11 years
• In those 11 years blood banking shifted from
whole blood storage to manufacture of
components

ADENINE 2 (CPDA 2)
• Beutler and West showed that packed RBC stored in
CPDA - 1 ran out of glucose sooner than whole blood
stored in CPDA 1.
• They suggested addition of more glucose to original
anticoagulant solution making CPDA -2
• This meant that plasma and platelets contained large
amounts of unnecessary sugar
• PRC stored in CPDA - 1 ran out of glucose very soon
compared to CPD
• CPDA -2 contains more glucose than CPDA-1
• Disadv: plasma and platelets loaded with sugar

CP2D
• 100% more glucose than CPD and 60% more
than CPDA-1
• The high glucose content of CP2D is necessary
because it is used with an additive solution
(AS-3)that does not contain sufficient glucose

CONTENTS
(mMol/L)
ACD-A ACD-B CPD CPDA-1 CP2D
CITRIC ACID 35 21 14 14 14
SODIUM
CITRATE
97 58 116 117 117
DEXTROSE 136 81 141 142 284
MONO
SODIUM
PHOSPATE
- - 15.8 16 16
ADENINE - - - 2 -
pH 5 5.6 5.6 5.6
VOLUME
RATIO USED
1:7 1:4 1:7 1:7 1:7
A/c to ROSSI’s

ADDED NUCLEOTIDE
ADENOSINE :
–ADVANTAGE: Restoration of ATP. This
was assumed to be due to its
phosphorylation to AMP and
subsequent conversion to ATP
–DISADVANTAGE :Adenosine was found
to have marked hypotensive effect. It
was never used due to its toxicity
GUANOSINE:
Used in PAGGS-M which provides 7 weeks of
RBC storage with recovery of 74 %
Guanosine has also been used as an additive
to ACD based blood preservative but
usefulness as practical additive has not yet
been established

INOSINE :
Adenosine deaminase rapidly converted adenosine to
Inosine
Inosine + P1  Ribose 1 phosphate (R-1-P) +
Hypoxanthine
R-1-P is readily converted
to F-6- P by pentose shunt
which feeds into glycolytic
pathway leading to
generation of ATP
ADVANTAGES:
Excellent effect on viability
DISADVANTAGES :
Hypoxanthine formed from inosine degraded
to uric acid
INOSINE can serve as indispensible part of
rejuvenation solution that are used to
revitalize red cells after they have lost their
ATP and 2,3 DPG

DIHYDROXYACETONE
• Introduced by Brake and Deindoefer in 1972
• Increased maintenance of 2,3 DPG levels
• MOA: RBC contain enzyme triokinase with
capability of phosphorylating DHA to DHAP.

OXALATE AND ASCORBATE
• Effect of Ascorbate on red cell 2,3 DPG
discovered in 1972- found to increase 2,3 DPG
levels
• Studies by Kandler et al show that pure
ascorbate has no effect at all. The oxalate that
contaminates it is responsible for increase in
2,3 DPG
• Oxalate precipitates renal calculi even in small
quantities , so its use not practical

XANTHONE DERIVATIVES
• Hyde et al,(1984) studied effect of tricyclic acid 2
ethoxy-6-5(tetrazolyl) xanthone (BWA825C) on O2
dissociation curve of Hb
• They found that it not only exerted an effect on
purified Hb solution but also increased 2,3 DPG
levels during storage
• Related compounds BWA440C and BWA827C
were also found to be effective in maintaining 2,3
DPG levels
• Little is known about the toxicity of these
compounds to permit their use clinically

INORGANIC PHOSPHATES
• Phosphate exerts multiple metabolic effects
on erythrocytes and when high concentrations
are added to blood preservatives ,levels of
both 2,3 DPG and ATP are enhanced
• Larger quantities have profound effect on ATP
preservation but do not have corresponding
advantageous effect on post infusion viability

PYRUVATE
• Pyruvate makes NAD available from NADH
through Lactate dehydrogenase reaction, the
addition of pyruvate to red cells may facilitate
metabolism through the glyceraldehyde
dehydrogenese step of glycolysis
• Pyruvate exerts a modest favorable effect on 2,3
DPG
• Pyruvate undergoes various polymerization
reactions and therefore unstable .Hence, little
practical use as component of blood preservative
• Used in Rejuvenating solutions

CITRATE
• Prevents coagulation by chelating calcium
• Retards glycolysis
• Side effects:
– Has affinity for magnesium ions-
hypomagnesaemia in the setting of massive
transfusion has been reported
– Citrate toxicity: in new born without adequate
calcium store and with premature liver
– Hyperglycemia is seen during massive transfusion
in orthotopic liver transplant
– In massive transfusion, citrate considered to be a
cause of cardiac arrhythmia
DEXTROSE
Improves red cell viability
Provides energy for ATP
synthesis
Decreases rate of hydrolysis of
phosphorus

SODIUM BIPHOSPHATE
• It acts as a buffer to control decrease in pH
from the generation of lactic acid
• Cells passively lose phosphate, so by adding
phosphate this loss can be reduced
The incorporation of adenine seems to
increase ADP levels thereby driving glycolysis
towards synthesis of ATP
As ATP-dependent cytoskeleton control red
cell membrane shape and rigidity , addition of
adenine to cells restores shape and post
transfusion viability .Adenine provides a
substrate for ATP synthesis in RBC resulting in
improved viability
Adenine approved by FDA in August 1978
ADENINE

CITRIC ACID
• Prevents glucose caramelization during
autoclaving
• Provides optimal pH with citrate for red cells

BLOOD : ANTICOAGULANT RATIO
• Volume of anticoagulant –nutrient solution is
normally 1/7 volume of collected blood
– 14 ml of CPD/CPDA is used in preserving 100 ml
blood
– 63 ml for a 450 ml collection
– 70 ml for 500 ml collection
• At the end of the collection , venous blood with
pH of 7.35 mixes with anticoagulant-nutrient
solution with pH 5.0 to 5.6 with resulting pH of
7.05 in the mixture

• ADDITIVE SOLUTIONS
• REJUVENATION SOLUTIONS
• RED CELL FREEZING

• The additive system concept was developed by Beutler in
the 1970s, and implementation of specific solutions was
initiated in the early 1980s by Lovric in Australia and
Högman in Sweden
• Additive solutions are preserving solutions that are added to
the RBCs after removal of the plasma with/without platelets
• Removal of the plasma component during the preparation of
RBC concentrates (high hemocrit preparations esp.) remove
much of the nutrients needed to maintain RBCs during
storage
• Removal of substantial amounts of adenine and glucose
present in anticoagulant-preservative solution lead to a
decrease in viability, particularly in the last 2 weeks of
storage
•Void of plasma with high hematocrits makes units to be
more viscous and difficult to infuse, especially in
emergency situations
•Additive solutions reduce hematocrits from around 70
to 85% to around 50 to 60%
•Additive solutions employed in the systems were
composed of standard ingredients used intravenously:
saline, dextrose, and adenine
•Additive solutions are of: First generation & second
generation
•First Generation consists of: AS-1, AS-3 ,AS-5 (USA) &
MAP(Japan )
•Second Generation consists of: BAGPM, only approved
are PAGGS-M, AS-7; PAGGGSM, ErythroSol-1,2 & 4

• Högman’s System: Standard CPD in primary bag
with the additive solution containing saline,
adenine, and glucose (SAG), further modified with
the addition of mannitol (SAGM), which protected
against storage-related hemolysis
• SAGM~ Adsol AS 1
• Lovric : doubled dextrose concentration in the CPD
(CP2D)and used it in connection with an additive
solution composed of saline, adenine, glucose,
trisodium citrate, citric acid & Na3PO4
• Formulations by Lovric and Högman provided the
basis for the three additive solutions (USA):
AS-1 (Adsol), AS-3 (Nutricel)& AS-5 (Optisol)

• AS-1 : SAGM (retard hemolysis) coupled with CPD (in
primary bag)
• AS-3 : SAG but at different concen. & in addition to
sodium phosphate, sodium citrate & citric acid ; AS-3 is
coupled with CP2D (in 10 bag)
• AS-5 : SAGM at different concen. and uses CPD in the
primary bag
• All of these additive solutions are approved for 42 days of
storage of RBCs
• None of the additive solutions maintain 2,3-DPG
throughout the storage time
• As with RBCs stored only with primary anticoagulant-
preservatives, 2,3-DPG is depleted by 2 weeks of storage
• Post storage survival rates of greater than 80 percent &
with less than 1 percent hemolysis.

• First Generation: to replace the volume and
sugar lost with plasma removal and add the
adenine necessary for storage beyond 3 weeks
• Mannitol reduced hemolysis and increased the
osmolarity of the solution further
• Second Generation: attempts to rebalance the
final suspending solution and a search for
additional nutrients for the packed RBC
concentrates
• BAGP was the result of the original attempt by
Beutler to preserve both ATP and 2,3-DPG by
raising the pH(resulted in high concen of 2,3 BPG
at the expense of ATP with no improvement in
storage conditions)

pH
>7.2
<6.4
Bifunctional enzyme diphosphoglycerate
mutase – phosphatase converts almost all
1,3-DPG into 2,3-DPG,depriving the cell of
new ATP
Activities of hexose kinase and
Phosphofructo kinase, are too low to
support ATP production
6.4-7.2
Hb, the mineral salts in the
suspension & bicarbonate all
serve to buffer the protons
produced by glycolysis

FIRST GENERATION
ADDITIVE SOLUTION
Phosphate (2mmol /100ml) in additive
solution buffers about 1mmol of
additional protons
Bicarbonate (2mmol /100ml) : protonated to
make carbonic acid, converted to CO2 and water
by red cell carbonic anhydrase, and buffer
2mmol of protons as the CO2 diffuses out of the
plastic bag

• Second Generation AS:
Its formulation and pH balance almost double
the amount of ATP energy available to stored
red cells by depressing diphosphoglycerate
mutase activity while sustaining glycolysis
• PAGGS-M : mannitol, the initials stand for
• phosphate, adenine, glucose, guanosine &
saline
• Guanosine: GTP was detected in red cells and
known to decrease during storage (may inhibit
primitive coagulation enzyme
transglutaminase)

FIRST GENERATION SECOND GENERATION
1. SAG 1. BAGP-M
2. SAG-M 2. PAGGS-M
3. AS-1 3.PAGGG-M
4. AS-2 4.Erythrosol-1
5. AS-3 5.Erythrosol-2
6.Erythrosol-81
7.EAS-64

Composition of Additive Solutions
CONTENTS
(per 100ml)
AS-1 AS-3 AS-5
Dextrose 2200 1100 900
Adenine 27 30 30
Monobasic sodium
phosphate
0 276 0
Mannitol 750 0 525
NaCl 900 410 877
Sodium citrate 0 588 0
Citric acid 0 42 0
Primary bag
anticoagulant
CPD CP2D CPD

BIOCHEMICAL CHARACTERISTICS
CONTENTS AS-1 AS-3 AS-5
Storage period
(days)
42 42 42
pH (at 370C) 6.6 6.5 6.5
24hr Survival (%) 83 85.1 80
2,3BPG (% initial) 68 67 68.5
Hemolysis (%) 0.5 0.7 0.6

PLATELET ADDITIVE SOLUTION
• PAS : Crystalloid nutrient media used in place of
plasma for platelet storage. They replace 60-70%
of plasma in platelet components, so the amount
of storage plasma can be decreased
• have a lower risk for allergic transfusion reactions
& TRALI
• PAS III received FDA approval
• Recovery range = 35% - 71%
• Survival range = 4.5 – 7.0 days

• The changes that lead to loss of viability by cumulative
oxidative damages occurring during storage are largely
reversible by a process called rejuvenation (metabolic
recharging of red cells at the end of their storage period)
• Hogman showed that rejuvenating red cells at the end of
six weeks storage in SAG-M increased their 24-hour in-
vivo recovery from 77 to 89%
• Initial rejuvenate solutions had phosphate, inosine,
glucose, pyruvate and adenine (PIGPA)
• This rejuvenation is a strictly metabolic recharging of red
cells at the end of their storage period
• They can be rejuvenated by incubation in a high-pH
solution of phosphate, inosine, pyruvate & adenine
(PIPA,Rejuvesol,etc) for 2 hours

• It increase the levels of 2,3-DPG and ATP in stored red
cells & can be added at any time between 3 days post
collection and 3 days after expiration of red cells and
increases in-vivo recovery of stored RBCs, probably by
allowing them to internalize negatively charged
membrane phospholipids that would otherwise signal
clearance by macrophages
• Return of the normal distribution of phospholipids also
prevents red cells from participating in plasma
coagulation reactions
• Rejuvenation does not reverse the oxidative damage to
band3 of the cell membrane, desialation of glycoproteins,
or loss of membrane

• 50ml solution is added directly to a unit of red cells,
mixed and incubated at 37 °C for one hour
• The rejuvenated red cells are either washed with saline
(2 Litres of unbuffered 0.9% NaCl) and can be kept at
2-6°C, however, they should be transfused with in 24
hours after washing or they are glycerolized for
keeping red cells in frozen state to improve the quality
of red cells
• Red blood cells rejuvenation solution, 50 ml sterile vial
(Rejuvesol) is commercially available; used primarily to
salvage liquid-stored RBCs (also autologous RBCs) that
have reached outdate
•Rejuvenated RBCs can be frozen with glycerol
as the cryo-protecting solution; rare units and
O-type units are primarily treated for
subsequent cryopreservation
•Because the processing including the washing
procedure is currently accomplished with open
systems that are not specifically designed to
prevent the entrance of bacteria, federal
regulations require that rejuvenated/ frozen
RBCs are used within 24 hours of thawing
•Rejuvenation process is expensive and time
consuming and is rarely used

RED CELL FREEZING
• Smith in 1950 reported that glycerol could prevent
freezing injury in human red cells and that red cells,
mixed with glycerol could be frozen without damage
• Glycerol, Dimethyl sulfoxide (DMSO) & Hydroxyethyl
Starch(HES) (cryoprotective agent) is added to red
cells they can be frozen and thawed without damage
by intracellular ice formation and hypertonicity
• Glycerol limits ice formation and provides liquid
phase in which salts are distributed as cooling
proceeds thereby avoiding excessive hypertonicity
• Frozen red cells are primarily used for autologous
transfusion and the storage of rare group blood
•Cryoprotective agent is added to red cells that are less
than 6 days old
• Glycerol (used commonly) is added to the red cells
slowly with vigorous shaking so that glycerol
permeates into the red cells
•The cells are rapidly frozen and stored in a freezer
•The freezing and storage temperature depends on the
concentration of glycerol.
•High concentration glycerol [40% weight in volume]
and a low concentration glycerol [20% weight in
volume] in the final concentration of cryopreservative
• Most blood banks use the high glycerol technique.

Characteristics High Glycerol Low Glycerol
Initial freezing
temperature
-80 °C -196 °C
Need to control
freezing
No Yes
Type of freezer Mechanical Liquid nitrogen
Maximum storage
temperature
-65 °C -120 °C
Shipping requirement Dry ice Liquid nitrogen

• Frozen cells are deglycerolized before transfusion
• Removal of glycerol is achieved by systematically
replacing the cryo-protectant with decreasing
concentrations of saline
• The cells are washed with 12% saline, followed by 1.6%
saline, with a final wash with 0.2% dextrose in normal
saline
• The shelf life of thawed red cells stored at 2-6 °C is 24
hours.
• The frozen red cells can be stored for 10 years
• The outdating period of the thawed red cells stored at
2-6°C is 24 hours.
• Red cells stored in additive solutions can be frozen up to
42 days

DMSO
• DMSO protects the cells by:
1) partially solublizing the membrane so
that it is less prone to puncture,
2) interrupting the lattice of the ice, so that
fewer crystals form.
• 10% concentration and is usually
combined with saline or serum albumin
• also used in the banking of
cord blood cells

DMSO TOXICITY
• DMSO's systemic toxicity is considered low
• It has effects on coagulation; anticholinesterase activity; DMSO-
induced histamine release by mast cells
• DMSO can decrease membrane thickening and induces
temporary water pores when used at low concentrations.
• Side effects of infusion of DMSO-cryopreserved cells include
nausea, emesis, chills, rigors, and cardiovascular events
• DMSO also shows neurotoxic effects including encephalopathy,
when stem cells were infused into cancer patients, as well as
gastrointestinal effects
• DMSO is also directly cell toxic, affecting cell viability, inducing
apoptosis and differentiation & decrease membrane thickening
and induces temporary water pores when used at low
concentrations. At higher concentration it induces disintegration
of the bilayer structure of the lipid membrane

HYDROXY ETHYL STARCH
• A synthetic modified polymer based on purified starch
• Large molecules of HES serve as a non-penetrating
cryoprotectant; this effect of depends on its ability to absorb
water molecules and keep these thermally inert in glassy state
without experiencing any phase transition during cooling
• HES influences the viscosity of solutions and decreases the
cooling rate required for optimal survival during vitrification,
increases propensity for supercooling and kinetically inhibits ice
formation (HES can absorb up to 0.5 g water per 1 g of HES)
• Accumulation of HES in the extracellular space, initially
increases extracellular viscosity reduces the rate at which water
can be withdrawn from the cells preventing osmotic stress and
damage
• HES has been used in the cord blood banking industry for the
separation of blood into its individual components

HES
• HES is widely used as a plasma volume
substitute due to its colloidal osmotic pressure
which increases viscosity of plasma and whole
blood and facilitates delivering of oxygen by
red blood cells, in hemodilution treatment to
enhance the microcirculation and for
peripheral arterial stenosis treatment
• (w/w) HES 6%,11.5% 12%,14% : used for
storage of RBC according to different studies

HES TOXICITY
• HES administration leads to increased serum amylase
concentration of up to 5 times the initial value.
However, this increase does not affect the pancrease or
lipase activity and therefore seems to have no
pathological relevance
• Osmotic nephrosis-like lesions in renal transplant
patients
• Pruritus on chronic administration
• bleeding complications due to decreased factor
VIII/von Willebrandt factor, platelet function defects
and incorporation into fibrin clots, probably due to
dilution effects
• Large HES molecules can cause detrimental effects on
rheological parameters of blood

MECHNISM OF CRYOPRESERVATION
Upon cryopreservation
• extracellular (1) and intracellular (2)
water crystallises to ice which can
lead to damage including to
membranes (3)
• DMSO opens cell walls and leads to
water being removed from the cell
(4) while DMSO enters
• HES in turn binds extracellular
water (5) and establishes a
concentration gradient which
removes water from the cell (6),
and can thereby confine ice
formation away from the cell (7)
• In addition, it is considered to
stabilize the cell membrane (8) but
normally without entering the cell

INDICATIONS FOR THE USE OF
FROZEN RED CELLS
• Freezing of rare blood groups enables long-term
storage and supply on a regional and national basis.
• Storage of blood for patients with antibodies against
high frequency antigens.
• Storage of blood for autotransfusion, specially in
patients with rare blood group.
• Prevention of non-haemolytic febrile transfusion
reaction in patients sensitized to leucocytes,
platelets or plasma protein.
• Prevention of sensitization against HLA antigens in
potential recipients of tissue transplants.

REFERENCES
• AABB TECHNICAL MANUAL 18TH EDITION
• ROSSI’s PRINCIPLES OF TRANSFUSION
MEDICINE 5TH EDITION
• www.mahasbtc.com/preservation-and-
storage-blood
• MODERN BLOOD BANKING & TRANSFUSION
PRACTICES (DENISE M HARMENING)
• Transfusion and Apheresis Science 46 (2012)
137–147

Anticoagulants

More Related Content

What's hot

Similar to Anticoagulants

More from Gayathri Nair

Recently uploaded

Anticoagulants