Review Article

Artifical Blood
Brig YK Goorha*, Maj Prabal Deb+, Lt Col T Chatterjee#, Col PS Dhot**,
Brig S Pra!ad, "SM++
Ab!tract
#li$i%atio% of &%'a%ted !ide(effect!, e!)eciall* tra%!f&!io%(tra%!$itted
di!ea!e! +,-" a%d he)atiti!. a%d le&coc*te($ediatedallo!e%!iti!atio%, i! a%
i$)orta%t goal of $oder% tra%!f&!io% $edici%e/ The )roble$! a%d high
co!tfactor i%0ol0ed i% collecti%ga%d !tori%g h&$a% blood a%d the )e%di%g
'orld('ide !hortage! are the other dri0i%g force! co%trib&ti%g to'ard! the
de0elo)$e%t of blood !&b!tit&te!/ T'o $ajor area! of re!earch i% thi!
e%dea0o&r are hae$oglobi%(ba!ed o1*ge% carrier! +,B2C!. a%d
)erfl&oroche$ical!/ #0e% tho&gh the* do %ot 3&alif* a! )erfect red blood cell
!&b!tit&te!, the!e 4o1*ge% carr*i%g !ol&tio%!5 ha0e$a%* )ote%tial cli%ical a%d
%o% cli%ical &!age!/ The!e ca% reach ti!!&e! $ore ea!il* tha% %or$al red cell!
a%d ca% deli0er o1*ge%directl*/ The!e are %ot 'itho&t ad0er!e effect!, a%d
e1te%!i0e cli%ical trial! are bei%g co%d&cted to te!t their !afet* a%d efficac*/
6e' &%der!ta%di%g! o% the $ode of actio% of the!e )rod&ct! 'ill hel) to
defi%e their &tilit* a%d a))licatio%/ 2%l* after !&cce!!f&lcli%ical trial! ca% the*
be &!ed for )atie%t $a%age$e%t, after a))ro0al b* the 7DA/
M8A7- 9::;< => ? @=(=:
Ke* Aord! ? Blood !&b!tit&te< ,ae$oglobi% !ol&tio%!< Perfl&orocarbo%!
main advantages include availability in large volumes,
storage for prolonged periods, rapid administration
(without typing and cross matching) and sterilization
There is a quest going on for a suitable bloodsubstitute, which can be stored on a
shelf, andinfused safely at any time and in any place, regardlessof the blood type.
Strictly speaking, infusion of anymaterial other than autologous blood, is actually
infusionof blood substitute. Thus, the history of bloodtransfusion can be regarded
as the history of bloodsubstitute. Substances like milk, casein derivatives,starch,
saline and inger!s solution had been tried priorto the first successful human to
human transfusion("lundell, #$%&). Since 'orld 'ar ((, the search for asuitable
alternative to blood has been intensified, toenable coping with war like situations
and large)scalecivilian disasters. The functions of each bloodcomponent and the
substitution of each are quite familiar,e*cepting that of o*ygen carrying capacity.
There hasbeen considerable progress in two ma+or classes ofsubstitutes viz.
haemoglobin solutions andperfluorocarbon (,-.) emulsions /#0.
,ae$oglob&li%(ba!ed o1*ge% carrier! +7ig B.
1on Stark (#$2$) was the first to use haemoglobin(3b) solution in the treatment of
a patient suffering fromanaemia. 4 lot of research has subsequently modified3b
solution to form purified 3b solutions. .urrently,3"5.s represent an interesting
class of bloodsubstitutes, which are undergoing advanced clinicaltrials. The
therapeutic goal of these compounds is toavoid or reduce blood transfusion in
different surgicaland medical situations of acute 3b deficiency. Theirmain
advantages include availability in large volumes,storage for prolonged periods,
rapid administration(without typing and cross matching) and sterilisation
bypasteurisation. Their main known disadvantages are,reduced circulation half)life,
haemodynamic andgastrointestinal perturbations, probably related to nitrico*ide
(65) scavenging, free radical induction, andalterations of biochemical and
haematologicalparameters (increase in liver enzyme levels, plateletaggregation) /#)
70.
P&rified hae$oglobi% !ol&tio%!
The first step in the pursuit for red cell substitute wasto produce 3b in a mass scale
and under sterile conditionefficiently, ensuring high purity but no denaturation. The
typical methods usedwerecrystallisation of stroma freehaemoglobin (S-3)
obtained by hypotonic haemolysis8
high performance liquid chromatography (3,9.)8utilising the high stability of
carbo*ylhaemoglobinagainst heating and dialysis (Somatogen .o) producedby
Escherichia coli /&08 and transgenic human 3bproduced by transgenic swine
(Swanson et al8 :6; .o)
/<0.
Modified hae$oglobi%
Pol*$eri!ed ,ae$oglobi%
(nitially, glutaraldehyde (a polymerising reagent forproteins) was used to develop
,9,)con+ugatedpolymerised 3b with an adequate o*ygen affinity /=0.Since the
number of molecules remained less, thecolloidal osmotic pressure could be kept
low (%> Torr,/3b0 ? #< gm@dl), thus enabling its transfusion as asolution with high
3b concentration. (t had a circulationhalf)life of %& hours and could be stored for
more than #year. The drawbacks were related to the distribution ofthe molecular
weight, uncertainty of the position ofreacted sites, and high viscosity of the
solution.
Transient renal tubular vacuolation after multiplein+ections have also been
reported. These problems weresolved by using chromatographic separation
techniques.(nitial trials were conducted on adult healthy males inthe Anited States,
Buatemala and in Caire /D0. (n #22%,the phase ( clinical testing of a modified
solution wascarried out by 6orthfield 9aboratories after approval ofthe -:4. The
ma*imum dosage tried was >.= gm@Eg(=7 gm ma*imum). 6o abnormality such
asvasoconstriction was noticed. (n the phase # trial usingpolymerised bovine 3b,
dosing of upto >.% gm@kg causeda rise in blood pressure and a decline in heart
rate,resulting in suspension of the trial /#0..urrently, soluble, cell)free, o)raffinose
cross)linkedand oligomerised human 3b (5)r)poly)3b)(3emolink,3emosol,
.anada)(molecular weight ranging from 7%)F<>> k:a) is in a phase ((( clinical
trial for peri)operativeuse in cardiac and orthopaedic surgery (at doses from%<g
(%<> ml)@#>> g (#>>> ml) /%,$0. (ts affinity foro*ygen appears lower than normal
blood and an n (3illcoefficient) value of about # indicates a very low degreeof co)
operativity. ,robably related to the low 5% affinityvalue and to the high molecular
weight, 5)r)poly)3bhas been shown to induce lesser haemodynamicperturbations
than other first generation modified 3bs.
Pol*$er Co%j&gated ,ae$oglobi% ?
This was produced by increasing the molecular weightof 3b, by bonding it with a
water)soluble polymer definedand distribution of the number of con+ugated ,5G
chains.Since this is a polymer)con+ugated type, viscosity is highand the colloidal
osmotic pressure is also as high as %&Torr, even at a 3b concentration of $ gm@dl.
The systemthat bonds bovine 3b may result in problems ofantigenicity on repeated
usage /20. 5ther con+ugated3bs like, de*tran)benzene)tetracarbo*ylate)
con+ugated3b (3b):e*)"T.), is undergoing animal trials /#>0.
-%tra$olec&lar Cro!!(Li%Ced ,ae$oglobi% ?
:iaspirin (7,<)dibromosalicyl fumarate8 :""-) wasused to cross)link, a)units and,
b)units of 3b, and formdiaspirin cross)linked 3b (:.9 3b). (t was thoughtthat
cross)linking would prevent its dissociation andincrease half)life during circulation
(mean half)life is#> hours). The viscosity of the solution is very low (%c,), and the
colloid osmotic pressure %$ Torr. 4t present,a production facility for mass yield is
in operation. ,hase( trial studies c#22% onducted by "a*ter in #22% have
revealedconsiderable success, e*cepting a dose)related elevationof blood pressure
and decline in heart rate, correspondingto ma*imum dosage of >.# gm@Eg /#0.
5!3ara et alobserved an increase in whole blood methaemoglobinfraction (>.$& H@)
>.DDI at baseline to &.>$ H@)#.7=I)during &$ hours of the :.9 3b infusion
(dosage J27=
H@)%D= mg@Eg), but it did not reach a range associatedwith complications /#>)#%0.
eco$bi%a%t ,ae$oglobi% ?
ecombinant 3b (r 3b #.#) is a method in which afew parts of an amino acid
sequence of human 3b are replaced to prevent the dissociation into dimers and
tomaintain adequate o*ygen affinity. The phase ( clinicaltrial in #22% recorded side
effects like fever, chill andheadache. "y improving on the purification process,the
endoto*in component was eliminated, thus reducingthe adverse effects. -urther
trials in #227, with ama*imum dosage of %<.< gm, recorded rise in bloodpressure
and mild gastrointestinal symptoms /#,&0. Thephase (( trials involved testing of a
newer variant ie.r3b &#&, where two r3b #.# were con+ugated to obtaina double
o*ygen carrying capacity and a five timesprolonged half)life per unit volume /#0.
,ae$oglobi% "e!icle! ?
The advantages of 3b vesicles are J #) no denaturationor modification of the 3b8 %)
made of purified 3b andlipids. To simulate 3b encapsulation within
".s,microcapsules made of nylon, collodion, gelatin, gumarabic and silicon were
tried, but they were rapidlyremoved by the reticuloendothelial system. (n
#2DD,:+ord+evich and Killer /#70 successfully used the theoryof phospholipid
vesicles for use in encapsulation andprepared 3b vesicles out of phospholipids,
fatty acids, cholesterol etc. /#0. ,resent research is focussed onliposome)
encapsulated 3b (9G3) sterilisation, controlof size, effective encapsulation and
stabilisation of thevessels /#%0. ecently a biodegradable polymermembrane)like
polylactide has been used to develop 3bnanocapsules /$0.
To1icit*
.ell)free 3bs, chemically altered or geneticallye*pressed in microbial host systems
(i.e. unmodifiedS-3), possibly cause e*cessive filtration of 3b dimersand tubular
obstruction leading to a decreased glomerularfiltration rate and renal damage. The
ma+or concernpertains to the interference of 3b and its o*idationproducts with the
vascular redo* balance, potentiallyimpeding its clinical usefulness. Gndoto*in
contaminants,residual cellular stroma, and lipid fragments are knownmediators of
to*icity leading to vasoconstriction,complement activation and generation of free
radicals
/#,#>,#&,#<0.
Perfl&orocarbo%! ?
,-. are chemically inert compounds consisting offluorine)substituted
hydrocarbons. Anlike 3b)based substitutes, ,-.s have the following advantages J
#)they do not react with o*ygen or other gases8 %) increasethe o*ygen solubility in
the plasma compartment8 7) the dissolved o*ygen is not sub+ect to the effects
oftemperature, p3, %,7):,B etc. (thus the o*ygendissociation curve is linear)8 and
&) facilitate effortlesstransfer of o*ygen from the red cells to the tissue. Sincethey
are insoluble in water, need for an emulsificationprior to intravenous use was felt.
Some workersadvocated use of albumin and lecithin, whereas Beyeret al used
,luronic -)=$, which is a mi*ture of shortchain polymers /#<0.
7ir!t ge%eratio% )erfl&orocarbo%! ?
The first commercially available ,-. was -luosol ):4 %>I (Breen .ross, 5saka,
Lapan). This used
,luronic -)=$, as an emulsifying agent, and was able tomaintain a balance between
the o*ygen carrying capacityand tissue retention. (t comprised two
,-.s,perfluorodecalin (,-:) and perfluorotrypropylamine (-T,4). ,-: was the
primary component and o*ygencarrier, whereas -T,4 was to provide the much
neededstability. Gach of the two components had different halflives,with ,-:
being only 7 to = hours, due to its rapidclearance. -T,4 on the other hand,
persisted in the tissuefor months /#<0.
Seco%d ge%eratio% )erfl&orocarbo%! ?
-luosol had paved the path for further refinement of,-.s. The desirable
characteristics in the secondgeneration ,-., as advocated by eiss and 9e "lanc
etal /#=0 were J #) large o*ygen)dissolving capacity, %)faster e*cretion and less
tissue retention, 7) lack ofsignificant side effects, &) increasing purity, and <)
largescale production and availability. The three candidateschosen as per these
criteria were ,-:, perfluorooctylbromide (,-5") and bis (perfluorobutyl)
ethylene.,-5", known in its emulsion as 5*ygent, was favouredfor clinical trials
because of its stability and highe*cretion rate.
9inear ,-.s like ,-5" dissolve o*ygen better thancyclic ones like ,-:. The
o*ygen solubility is inverselyproportional to the molecular weight and
directlyproportional to the number of fluorine atoms. 2>Iweight@volume
emulsions of ,-5" have the capacity tocontain four times the amount of o*ygen
as compared toa first generation ,-.. ,-.s being chemically inert,are not
metabolised but are removed from the circulation,within &)#% hours, by the
reticuloendothelial system.They are stored in the liver and spleen and
subsequentlye*haled through the lung. educing the particle size orincreasing
theconcentration leads to increased tissuedeposition and subsequent tissue damage
/#<,#D0.
To1icit*
The ma+or problem with the first)generation ,-.swas due to complement
activation. 4n in)vitrocomparison of lecithin)based ,-: emulsions andpluronic)
based ,-: emulsions, showed the latter to bevirtually non)to*ic to peripheral
human leukocytes,e*cept causing mild to moderate inhibition of
endoto*ininducedcytokine production. 9ecithin)based ,-:emulsion caused
substantial cytoto*icity in phagocyticcells like monocytes (=>)#>>I after %& hour
incubation)and granulocytes (#>)%>I after %& hour incubation).They also
suppressed endoto*in)induced cytokineproduction in monocytes to more than 2$I
and inhibitedcell proliferation of an endothelial (G.1 7>&) and amonocytic cell
line (KonoKac=) to more than 2<I /#$0.3owever, an in)vivo trial of pluronic -)
=$ ) based ,-:led to adverse effects like intolerance of initial test dose,transient
leucopenia, hypotension, chest pain, and asyndrome consisting of fever,
leucocytosis and infiltrateson chest roentgenogram. (n some cases there was
aninitial reaction to the test doses.4cute to*icities due to the more refined
secondgeneration,-.s, were in the form of transient facialflushing, backache,
fever and a flu)like symptom within# to & hours post)infusion. These effects were
attributedto the normal clearance of ,-.s by the reticuloendothelialsystem.
5psonisation and phagocytosis of ,-. dropletsleads to activation of macrophages
andrelease of prostaglandins and cytokines by the archidonicacid pathway /#<,#20.
.hronic or delayed side effectswere characterised by inhibition of platelet
aggregationwith 2>I weight@volume ,-5" emulsion, and
transientthrombocytopaenia (not below $>,>>>@Ml). 5wing to itsorgan retention
effect, temporary histologic changes likeenlargement and appearance of vacuolated
histiocyteshave been noted in liver biopsies /#<,%>0.
Pote%tial cli%ical &!e
.onventionally, it is recommended that, forhaemorrhages less than =>> ml, plasma
e*panders shouldbe used8 for =>>)#%>> ml red blood cell products arenecessary8
and for blood loss above this, transfusion ofplasma derivatives and @ or platelet
products, or wholeblood is vital. esearch with blood substitutes conductedon
animal model and isolated human trials, recommendsits usage to treat
haemorrhages where blood loss isbetween =>>)#%>> ml. (t should be used as an
alternativeand@or supplement to homologous and autologoustransfusion, or in
combination with the use oferythropoietin /#<0. 1arious clinical, non)clinical
andparado*ical utilisations have been envisaged (Table #).
Table #
Pote%tial cli%ical a))licatio%! of o1*ge% carr*i%g !ol&tio%!
#. Therapy
(a) "lood substitutes J hemorrhagic shock8 hemorrhage (war,surgery)8 anaemia.
(b) 'hole)body rinse out J acute drug into*ication8 acute hepaticfailure.
(c) 9ocal ischemia J acute K(8 evolving K(8 cardiac failure8 braininfarction8 acute
arterial thrombosis and embolism8 ,T.4 ofcoronary artery.
(d) Beneral ischemia J gas embolism8 .5 into*ication8 34,5834.5.
(e) 4id for organ recovery J acute renal failure8 acute hepatic failure8acute
pancreatitis.
(f) (nfectious disease J anaerobic and aerobic diseaes8
(g) 4d+uvant therapy J tumour radiotherapy8 chemotherapy
%. ,erfusional protection of organs during surgery ) cardiopulmonary
bypass, deep hypothermia, circulatory arrest, cardioplegia.
7. ,reservation of donor organ.
&. :rug carrier ) drug)con+ugated haemoglobin and perfluorochemicals.
<. .ontrast agent ) (,erfluoro)octylbromide)
6o%(Cli%ical A))licatio%!
#. .ulture medium
%. .hemical e*amination ) o*ygen sensor8 standard solution for o*ygen
calibrator
7. "ioreactor
,arado1ical Dtili!atio%! +of high(o1*ge% affi%it*.
#. 5*ygen absorbent
%. 5*ygen pulse therapy for malignant tumour in combination with
radiotherapy or chemotherapy..linical Ase J The efficacy of 3b solutions
inmanaging haemorrhagic shock and anaemia with lowhaematocrit, is better than
crystalloid and colloidsolutions. These have been used to preserve isolatedorgans,
as cardioplegic agent, and as ad+unct topreoperative autologous donation. 4 trial of
bovine 3bsolution in children with sickle cell anaemia, showedclinical
improvement without any adverse effects. (t hasbeen tried in cases of organ
recovery during organfailure, in infectious diseases, and as ad+uvants in
tumourtherapies. ,oly3b, :.93b and recombinant 3b have been used in clinical
trials of trauma patients /#<,%#,%%0.
"ased on the effect of preserving aerobic metabolism during ischaemia and
improving peripheral circulationby the o*ygen carrying solution, these agents
have beentried for perfusional protection of organs, 3b molecules and
perfluorochemicals can be used to reach the blockedcapillary beds, while
increasing the drug sensitivity ofthe peripheral tissues by o*ygenation.
,erfluorooctylbromide can be used as a new contrast agent witho*ygen carrying
capability in ultrasound, .T scan,angiography, K(, liver and spleen imaging and
tumourimaging /$,%%)%<0.
6on clinical applications J The o*ygen carryingsolution can be used in culture
media of the tissues andbacteria (aerobes)8 in chemical e*amination such as
anindicator for o*ygen sensor, and a standard solution foro*ygen calibrator8 and in
some kinds of bioreactor /%<0.,arado*ical utilisations J (f the o*ygen affinity of
theartificial red cells can be controlled, they can be used asan o*ygen absorbent
agent parado*ically. This can pavethe way for a new form of tumour therapy ie.
No*ygenpulse therapy combined with radiotherapy andchemotherapyO. This would
be in two stages. (n thefirst stage, Nlow o*ygen affinity solutionO, will be usedas an
ad+uvant therapy of radiation to deliver the o*ygento the tumours to increase
radiosensitivity. The secondstage would comprise Nhigh o*ygen affinity
solutionO,namely o*ygen absorbent, delivered selectively to thefeeding artery of
the tumour e*pecting ano*ic necrosisof the tumour /%<0.
Pre!e%t !tat&!
ed cell substitutes are being developed for use inblood replacement therapies,
either for perioperativehaemodilution or for resuscitation from haemorrhagicblood
loss /%=0. There is an urgent need for theseproducts because of risks associated
with bloodtransfusions and pending worldwide blood shortages.
esearch in this field has benefited from developmentof new technologies in
protein engineering. .linical trialsare being conducted to ascertain the safety and
efficacyof these newly developed products /#,%,D,#<0. 4lthoughcross)linked and
polymerised products have been foundto be safe in preclinical and early phase (@((
clinical trials,they have had difficulty in proving efficacy. The primaryadverse
effect for the ma+ority of cross)linked orpolymerised products is a haemodynamic
response,leading to increased vascular resistance to blood flow.
Artificial Blood @>
The physiological mechanisms are still incompletelyunderstood, so that safety and
efficacy cannot be
completely dissociated. 6ew understandings on the modeof action of these
products will help to define their utilityand application /%D0. 5nly one product, a
,-.compound, -luosol (Breen .ross, 5saka, Lapan), hasgained regulatory
approval for limited use. .onsiderableprogress has been made in developing
platelet substitutesalso. 9yophilised platelet, infusible platelet membranes,red cells
bearing arginine)glycine)aspartic acid ligands,fibrinogen)coated albumin
microcapsules and liposomebasedagents have been developed as putative
alternativesto the use of allogeneic donor platelet, to augment thefunction of
e*isting platelets and@or provide aprocoagulant material capable of achieving
primaryhaemostasis in patients with thrombocytopenia /%$0.
Co%cl&!io%
The final goal of any transfusion service is to createa transfusion system with no
side effects and with moreeffective medical care. The current system
ofhomologous blood, although is marred by manyproblems, like allosensitisation
and transfusiontransmittable diseases, is working well with low cost,acceptable
efficacy and relatively less side)effects.Gven so, the technologies of the future
promise togenerate an effective blood substitute, which willdefinitely have an
impact on transfusion medicine andthe transfusion services. ,resently, the prospect
of usingartificial blood is difficult to envision owing to problemsof short half life,
prolonged tissue retention, potentialto*icity, and issues of cost, cost relative
benefits,availability of raw materials and strict -:4 regulations.This may cause a
temporary shift of focus fromcompletely replacing blood cells, to that of using this
asan ad+unct to homologous transfusion. 3owever, in prehospitalor battlefield
resuscitation, or in countries wheresupply of safe blood is affected by potential
threat of3(1 infection, the role of blood substitutes willundoubtedly be of
immense value. 4nd it is absolutelycertain that a new system of blood service with
artificialblood and blood substitutes, including artificial o*ygencarriers and
recombinant plasma components, will bedeveloped in the near future, which will
confer a newdimension to transfusion medicine.