Presented by : Dr. Prakriti Shukla

Red blood cell substitutes The term “blood substitute” was first used for

plasma expandersLater for blood components

Presently it is applied to ex-vivo produced therapeutic materials with potential to replace or

reduce transfusion requirement of blood and blood components

Need for the development1. With rapid advances in surgical sophistication and increasingly

aggressive protocols of cancer management in the recent past ,the transfusion requirement of red cells have outgrown human resourse.Transfusion alternatives and blood substitutes are necessary to narrow the gap between demand and supply.

2. Risk of transmissible infections with blood transfusion has occupied frontline attention. Blood substitutes which undergo vigorous in vitro sterlization can alleviate both risk and cost in this regard.

3. Sudden transfusion requirements triggered by massive blood loss in accidents, natural disasters etc. cannot be entirely met by allogenic blood transfusions. Off – the –shelf available blood substitutes can be life saving.

Need contd..

4. Blood substitutes that are devoid of immune identity markers are universally acceptable that help to cut across all immunogenic risks of allogenic transfusions like incompatibility reactions, graft versus host reactions, allergic and febrile reactions, and transfusion related lung injury.

5. Limited shelf life of allogenic components resulting in wastage beyond the expiry date can be avoided by blood substitutes that show promise of longer shelf life even for several years if stored in a lyophilized state.

2 main indications for the transfusion of red cells : 1. severe haemorrhage2. chronic symtomatic anaemia for which no specific therapy

exist.

In both the circumstances , the aim of red cell transfusion is to improve the oxygen supply to the tissues by raising the oxygen content of the blood , according to the equations:

Development of red cell substitutesAs a result of massive research and

competition in pharmaceutical companies, three categories of red cell substitute have emerged

Cell free haemoglobin solutionsThe most important function of red blood cell is

to carry oxygen and carbon dioxide by virtue of its haemoglobin content .Therefore , an alternative to red blood cell transfusion , RBC free haemoglobin solutions have been prepared.

Problems encountered :1.Outside the RBC, the nomal tetrameric Hb

molecule undergoes fragmentation into dimeric forms which have following effects:

2. Outside the red cell free Hb looses its natural buffering environment of 2,3 DPG thereby adversely affecting the binding and release of O2 by free Hb molecule.

3. The Intraerythrocytic environment provides for the important reductive enzymes and mechanisms which combat the very oxidative nature of oxyhaemoglobin.

4. RBC free Hb is devoid of red cell enzymes and are therefore incompetent to prevent reperfusion tissue injury.

Strategies to overcome above

difficulties -1. Modified Haemoglobins a) polymerized Hb (cross –

linked) b) conjugated Hb2. Recombinant Haemoglobins3. Encapsulated Haemoglobins4. Perfluoro chemical emulsion

1. Cross linked hemoglobinCross linked hemoglobinTo produce cross-linked hemoglobin, small bridges of sugar molecules are covalently attached to the dimers to create a stable tetramer.

2. Polymerized hemoglobinPolymerized hemoglobinTo polymerize hemoglobin, surface amino acid

groups are linked by reagents such as glutaraldehyde.

Polymerized hemoglobin is the only product to date that has not triggered significant vasoconstriction after infusion.

3. Surface-modified hemoglobin3. Surface-modified hemoglobinSurface-modified hemoglobin is created by

attaching large molecules, such as polyethylene glycol, to surface lysine groups.

This modification also increases the viscosity and oncotic pressure of the solution.

4. Perfluorocarbon-based substitutes

PFCs are synthetic hydrocarbons with halide substitutions and are about 1⁄100th the size of a red blood cell.

These solutions have the capacity to dissolve up to 50 times more oxygen than plasma.

PFC solutions are modified hydrocarbons,however, they do not mix well with blood and must be emulsified with lipids or oils.

The best results are obtained if the patient is breathing 100% oxygen at the time of infusion(PaO2 ≥ 350 mm Hg).

Oxygent- The product is currently in phase III clinical trials for use in cardiac and general

surgical patients Fluosol (Japanese) - withdrawn c/o

complement activation & side effectsPertorfan - licensed in Russia and Mexico

New Perfluoro compound !!!POLYFLUORO-OCTOBROMIDE (PERFLUBRON)POLYFLUORO-OCTOBROMIDE (PERFLUBRON)This new perfluoro compound, which is

radioopaque, has two advantages over its predecessors.

First, higher concentrations of perfluoro compound may be administered, because a 100% (w/v) emulsion with phospholipid has a sufficiently low viscosity to be infused without dilution.

Second, oxygen is more soluble in polyfluoro-octobromide than in anyother perfluoro compound introduced to date

5. Microencapsulated haemoglobin

An alternative to polymerisation of haemoglobin to keep it for longer in the circulation is to incorporate it in a lipid membrane or liposomes which will act as “pseudoerythrocytes”

This prevents the escape of the molecule through the glomeruli, and the oxygen affinity of the solution containing the microspheres is similar to that of whole blood;

If pyridoxine 5-phosphate is added to the haemoglobin first a stable product is created.

The half life in the circulation is about 20 hours, which is similar to that of a polymerised haemoglobin solution.

6. 6. Recombinant haemoglobin/ genetically Recombinant haemoglobin/ genetically engineered engineered

haemoglobinhaemoglobinIt It is obtained by inserting the gene for human

hemoglobin into bacteria and then isolating the hemoglobin from the culture.

This process allows for the manipulation of the gene itself to create variant forms of hemoglobin.

One unit of hemoglobin solution can be produced from 750 L of Escherichia coli culture.

Optro - recombinant human Hb ( -linked)developed by Baxter (discontinued)

Hemoglobin Based Blood Products: Current Status

Hb Source TreatmentHb Source Treatment ProductProduct CompanyCompanyHuman Hb, outdated blood, Human Hb, outdated blood, x-linked x-linked

HemHemAssistAssist

BaxterBaxter

Bovine Hb, heterogeneous Bovine Hb, heterogeneous MWMW

Hemo Hemo PurePure

BiopureBiopure

Human Hb, outdated blood, Human Hb, outdated blood, x-linked, polymerizedx-linked, polymerized

HemolinkHemolink HemosolHemosol

Human Hb, outdated blood, Human Hb, outdated blood, chemically polymerizedchemically polymerized

PolyHemePolyHeme NorthfielNorthfieldd

Genetically engineered HbGenetically engineered Hb OptroOptro SomatogSomatogenen

Ideal red cell substitute

Easily picks up oxygen in the lungsEasily delivers oxygen to the tissuesDoes not need crossmatchingLong shelf lifeStable at room temperature

How Far How Near !!Different materials are being used to encapsulate hemoglobin, one

being a biodegradable polymer called polyactide.

Incorporating nanotechnology : An example is the use of dendrimer polymers to transport dissolved oxygen via intramolecular void spaces and on its surface

Exploring other carriers: other oxygen carriers such as hemerythrin are being explored-less prone to oxidative and nitrosative stress which would alleviate problems like NO scavenging and methemoglobin production

Applying HBOCs in other areas : cross-linking hemoglobin with tyrosinase which can increase the efficacy of chemotherapy and radiation therapy in tumor tissue

Unable to duplicate immunological and clotting properties of blood

Hemopure is one of the farthest along Prior preparation not required Stable for three years at room temperature Reported compatible

with all blood groups

TAKE HOME MESSAGE !!!The red cell substitutes have far too short a

survival time in circulation ,therefore, it is much easier to envisage a role in short term procedures such as immediate resuscitation of military or civilian casualties.

Avoidance of allogeneic blood transfusion would not only be beneficial for the individual patient, but if the practice became widespread it would help relieve pressure on transfusion services for ever increasing quantities of allogenic blood

THANK YOU !!