BLOOD SUBSTITUTES

By

MB ChB, FRCA Visiting Instructor in Anesthesiology and Critical Care Medicine

Lisa Milligan

Why do we need blood substitutes?

Blood shortages
Public opinion on virus transmission / life insurance issues Aging population Advancements in surgical procedures

HIV New blood-borne diseases, eg nvCJD Cost of safe transfusion in developing world

Problems associated with blood transfusion

Blood shortages & donor recruitment Compatibility need for cross-matching Cost of blood processing Shelf-life & storage Human error Unnecessary transfusions Risk of disease transmission Cultural & religious objection

Risks of disease transmission

Risk factor Estimated frequency Deaths per million per blood unit units of blood transfused 1 in 250,000 1 in 1,000,000 1 in 3,000,000 1 in 4,000,000 1 in 5,000,000 <0.5 <0.5 <0.5 <0.5

Hepatitis B Hepatitis C HIV Bacterial contamination

Blood Substitutes
Blood substitutes are fluids which when injected into the human blood stream contribute significantly to the transport of oxygen around the body
Cell-free oxygen carriers Oxygen therapeutics Red cell substitutes

How would a blood substitute be used?

Coupling with autologous blood Supporting transfusion service in developing countries Battlefield or natural disasters Alternative to blood transfusion for patients with religious objections

Properties of an ideal blood substitute

Adequate oxygen uptake in the lungs Adequate oxygen delivery to the tissues Long circulation time Non-toxic Rapidly excreted without causing harm Stable at room temperature Easily sterilized Cheap to manufacture Long shelf-life & easy to store Widely applicable w/o cross-matching Free of side-effects

Types of Blood Substitute

Biometric mimics natures way of delivering oxygen to the bodys tissues, e.g. Hb based substitute Abiotic use of totally synthetic chemicals to deliver oxygen to the tissues, e.g. PFC based substitiute

Types of Blood Substitutes

Hemoglobin Based Oxygen Carriers (HBOCs)

Bodys natural O2 transporter Complex protein consisting of 4 subunit chains: 2 alpha and 2 beta Each subunit contains an iron atom, which binds oxygen reversibly Inside the RBC Hb exists in a stable environment containing the enzymes it requires to control O2 binding & other characteristics

O2 Delivery by hemoglobin

Sources of Hemoglobin
Hemoglobin Human Blood Explanation
Involves extraction of Hb from donor blood Uses Hb from cow blood

Advantages
Cheap; uses discarded blood

Disadvantages
Supply of waste blood diminishing. Unacceptable to JWs Unknown long-term effects. BSE cows & CJD High production costs

Cow Blood

Cheap, plentiful. Less chemical modifications. Acceptable to JWs Infinite supply. Avoids human blood. Pure, virusfree Hb Potential infinite supply of large quantities of Hb

Microorganisms Transgenic

Genetically-modified bacteria & some plants can be made to produce Hb Genes for human Hb inserted into a developing animal

Ethical objections to Hb factories. Hb extraction difficulties

Cell-free hemoglobin

Characteristics of HBOCs
Size Microvascular effects Vasoactivity O2 Affinity (P50) Oxidation Absence of pro-inflammatory properties

Size
64 kDa Hb tetramer dissociates into and dimers Filtered through renal glomerulus & disappear from circulation w/i few hours Nephrotoxic Prolong T1/2 (12 36h) & minimize nephrotoxicity by:
Stabilization of the tetramer Polymerization of tetramers to oligomers Surface conjugation to MW and diameter

Elimination then by RES

Microvascular effects
Hb solutions: low viscosity, high oncotic pressure Low viscosity shear on endothelial cells vasodilators (endothelin & prostacyclin) local vasoconstriction & regional blood flow Hamster skin fold model Hemopure (polymerized bovine HBOC) local tissue PO2 compared with NS or Dextran

Vasoactivity
Many HBOCs have systemic pressor effect Pulmonary hypertension (in animals) Mechanisms not fully understood Free Hb closer to endothelium, binds nitric oxide & produces vasoconstiction Greater vasoconstriction with lower MW products Stimulate catecholamine release from adrenal medulla & potentiate response to norepinepherine endothelin-1 levels vasoconstriction cardiac output Sheep model of intra-op hemorrhage - HBOC produced better volume expansion than RL, more rapid MAP, CO in recovery phase, no DO2

Oxygen affinity (P50)

Hb outside of RBC loses its 2,3-DPG Affinity for O2 (P50 ) left-shift of oxyhemoglobin dissociation curve Reversal of left-shift attempted by pyridoxylation or Cl May be desirable property: HBOCs with low O2 affinities which unload O2 at higher PO2 , may trigger autoregulatory local vasoconstriction & impaired O2 delivery

Oxidation
In RBC Hb protected from oxidation by methemoglobin reductase In absence of enzyme, Hb auto-oxidizes to methemoglobin, which does not carry O2, and further metabolites including ferryl radicals HBOCs under development have low levels of methemoglobin (<3% - <15%) DCLHb in sheep peak methemoglobin of 5-7%, producing small O2 sat Polymerized bovine HBOC in humans peak methemoglobin 3.7% at 3 days post-infusion (not physiologically significant)

Absence of Pro-inflammatory Properties

Plasma from banked RBCs stored > 14 days accumulates pro-inflammatory substances which can produce SIRS HBOCs lack ability to activate WBCs in vitro Trauma patients resuscitated with PolyHeme showed no evidence of neutrophil priming in vivo

Modified - hemoglobins

Cross-linked HBOCs
HemAssist (Baxter) stabilized hemoglobin tetramer by diaspirin linkage Stored frozen Phase III trials halted due to safety concerns (worse outcome in stroke & trauma patients) Similar outcome in CPB compared with RBCs Animal TBI + hemorrhage CO, MAP, cerebral O2 saturation Somatogen (Optro, Baxter) recombinant human hemoglobin cross-linked by single polypeptide consisting of 2 subunits, joined by shorter linker peptide to 2 conventional -chains Development discontinued during Phase I and II trials (hypertension)

Polymerized HBOCs
PolyHeme (Northfield) glutaraldehyde polymerized human hemoglobin; pyridoxylated & extensively purified Trauma patients who received PolyHeme required fewer transfusions of banked blood Case report: MVA-victim JW 5U PolyHeme for severe hemorrhage (Hb 3.2g/dL) sustained for several days until hemorrhage controlled & erythropoesis stimulated by EPO compensated for blood loss Hemopure (Biopure) glutaraldehyde polymerized bovine Hb. Used as peri-op bridge Slight pressor effect & CI AA repair 27% receiving Hemopure avoided PRCs (cf none of controls) Licensed for clinical use in South Africa Vetinary use FDA approved (Oxyglobin) US Phase II on hold Hemolink (Hemosol) polymerised human hemoglobin using oxidised trisaccharide, O raffinose followed by reduction step Mild pressor effect Phase II trials in dialysis and ANH Cardiac surgery fewer transfusions at 5 days cf pentastarch Phase III trials completed in Canada, US & Europe. Development discontinued due to MI

Conjugated HBOCs
PEG-Hemoglobin (Enzon) polyethylene glycol conjugated to bovine Hb tetramer Much larger molecular radius Longer T1/2 than most HBOCs (48h) Hyperoncotic Used as sensitized for radiation treatment of solid tumours PHP (Apex Bioscience) pyridoxilation of human Hb followed by conjugation with polyoxyethylene Hypertensive effects Phase III trials in septic & hemorrhagic shock Hemospan (Sangart) human Hb tetramer conjugated to polyethylene glycol Larger molecular diameter, high viscosity & high O2 affinity to minimize autoregulatory & vasoconstrictive effects Phase III trials

Perfluorocarbon based substitutes

Perfluorocarbons
Perfluorocarbons (PFCs) are organic compounds similar to hydrocarbons - fluorine, rather than hydrogen atoms. Clear, odourless fluids, chemically very unreactive; linear, cyclic or polycyclic. The stability of PFCs stem from the strength of carbon-fluorine bonds. Also responsible for the inert nature of PFCs in the bloodstream. 2 most commonly uses PFCs are:
Perfluorodecalin (Flusol and Perftoran) Perflubron (Oxygent)

Perfluorocarbons
Synthetic liquids which dissolve large quantities of O2 Also transport CO2, N2 O2 easily extracted at tissues Stable, no chemical modification required, chemically inert Blood half-life dose dependent and limited by uptake by RES, eventually excreted from body by exhalation Easily sterilzed, no disease transmission Low production costs, infinite supply

Gas carriage by PFC emulsions

Microvascular effects of PFCs

Emulsion particles 0.2m diameter perfuse smallest capillaries (4 - 5m diameter) where no RBCs flow Augment local O2 delivery much more than would be expected from in O2 content of arterial blood O2 in dissolved state higher PO2 in microcirculation driving pressure for diffusion of O2 into tissues O2 transported by PFCs is preferentially metabolised due to its excellent unloading characteristics

Problems with perfluorocarbons

Immiscible with plasma, need to be prepared as emulsions (egg yolk phosphatide) Require high FiO2 to dissolve adequate quantities of oxygen; limits applications to places where supplementary O2 can be provided Flu-like symptoms observed in human clinical trials, delayed febrile reactions (due to phagocytosis by RES) Thrombocytopenia at higher doses (no effect on coagulation or bleeding time)

Perfluorocarbon products
Fluosol-DA (Green Cross Corporation, Japan) Acute hemorrhage in patients who refuse blood transfusion for religious reasons; performance disappointing Approved for use following PTCA but cumbersome & low efficacy Oxygent (Alliance Pharmaceutical Corporation, San Diego) ANH in dogs CO, mixed-venous PO2 & Sat Near-fatal hemorrhage in pigs mortality (43% 13%) Dogs undergoing CPB increased survival Reduced transfusion requirements in orthpedic & urologic surgery Development on hold due to safety concerns (stroke) Oxyfluor (HemaGen/PFC, Waltham, MA) Discontinued due to safety concerns

Current status of Blood Substitutes

Product class Product
Perfluorocarbons Oxygent Oxycyte Oxyfluor Cross-linked Hb HemeAssist rHb1.1 rHb2.0 Polymerized Hb PolyHeme HBOC-201 (Hemopure) Hemolink Conjugated Hb PHP PEGHaemoglobin Hemospan

Company
Alliance Synthetic Blood HemaGen Baxter Somatogen Baxter Northfield Labs Biopure Hemosol Apex Bioscience Enzon Sangart

Technology
PFC Emulsion PFC Emulsion PFC Emulsion Cross-linked Hb Recombinant Hb Recombinant Hb Glutaraldehyde, pyridoxal Hb Glutaraldehyde bovine Hb Polymerized Hb PEG-human Hb PEG-bovine Hb PEG-human Hb

Status
On hold; safety (stroke) Phase II Discontinued; safety Discontinued; safety (increased mortality) Discontinued; safety (hypertension) Discontinued; safety Phase III (enrolling) US phase II on clinical hold Discontinued; safety (MI) Phase III septic shock Discontinued Entering Phase III

Research Developments
Hemoglobin from worms: Lumbricus terrestris, Arenicola marina Hb polymers, 50x larger than human No modification required to remain stable in bloodstream long enough to oxygenate tissues No breakdown and kidney damage Pre-clinical testing in mice: normal O2 carrying capacity & no allergic reactions ?ease of extraction & purification in sufficient quantities ?hypertension

Synthetic Red Blood Cells

Encapsulation of hemoglobin in biodegradable polymer membranes Stabilizes Hb - prevents breakdown & kidney problems Trap natural RBC enzymes with Hb, creating a microenvironment for the Hb similar to normal blood Ensure correct O2 and nitric oxide binding & release Avoid hypertension Micro-organisms, such as bacteria & fungi, will be used to produce novel heme proteins