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BLOOD SUBSTITUTES

By
By

Lisa Milligan

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

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

• Human error • Unnecessary transfusions • Risk of disease transmission • Cultural & religious objection

Risks of disease transmission

Risk factor

Estimated frequency per blood unit transfused

Deaths per million units of blood

Hepatitis B

1

in 250,000

<0.5

– 1 in 1,000,000

Hepatitis C

1 in 3,000,000

<0.5

HIV

1 in 4,000,000

<0.5

Bacterial

1 in 5,000,000

<0.5

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 nature’s way of delivering oxygen to the body’s 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

Types of Blood Substitutes

Hemoglobin – Based Oxygen Carriers (HBOCs)

Hemoglobin – Based Oxygen Carriers (HBOCs) • Body’s natural O 2 transporter • Complex protein consisting

• Body’s 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

O 2 Delivery by hemoglobin

Sources of Hemoglobin

Hemoglobin

Explanation

Advantages

Disadvantages

Human Blood

Involves extraction of Hb from donor blood

Cheap; uses discarded blood

Supply of waste blood diminishing. Unacceptable to JW’s

Cow Blood

Uses Hb from cow blood

Cheap, plentiful. Less chemical modifications. Acceptable to JW’s

Unknown long-term effects. BSE cows & CJD

Micro-

Genetically-modified bacteria & some plants can be made to produce Hb

Infinite supply. Avoids human blood. Pure, virus- free Hb

High production costs

organisms

Transgenic

Genes for human Hb inserted into a developing animal

Potential infinite supply of large quantities of Hb

Ethical objections to ‘Hb factories’. Hb extraction difficulties

Cell-free hemoglobin

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 α and β 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, COin 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

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 α subunits, joined by shorter linker peptide to 2 conventional β-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

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:

PFCs in the bloodstream. • 2 most commonly uses PFCs are: – Perfluorodecalin (Flusol and Perftoran)

– 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

Gas carriage by PFC emulsions

Microvascular effects of PFCs

• Emulsion particles 0.2μm diameter perfuse smallest capillaries (4 - 5μm 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 it’s 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

Company

Technology

Status

Perfluorocarbons

Oxygent

Alliance

PFC Emulsion

On hold; safety (stroke)

Oxycyte

Synthetic Blood HemaGen

PFC Emulsion PFC Emulsion

Phase II Discontinued; safety

Oxyfluor

Cross-linked Hb

HemeAssist

Baxter

Cross-linked Hb

Discontinued; safety (increased mortality) Discontinued; safety (hypertension) Discontinued; safety

rHb1.1

Somatogen

Recombinant Hb

rHb2.0

Baxter

Recombinant Hb

Polymerized Hb

PolyHeme

Northfield Labs

Glutaraldehyde, pyridoxal Hb Glutaraldehyde bovine Hb Polymerized Hb

Phase III (enrolling)

HBOC-201

Biopure

US phase II on clinical hold

(Hemopure)

Hemolink

Hemosol

Discontinued; safety (MI)

Conjugated Hb

PHP

Apex

PEG-human Hb PEG-bovine Hb

Phase III septic shock Discontinued

PEG-

Bioscience

Haemoglobin

Enzon

   

Hemospan

Sangart

PEG-human Hb

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

& no allergic reactions • ?ease of extraction & purification in sufficient quantities • ?hypertension
& 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