Blood Substitutes

Roy WangEllen Quach

November 2005

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Why Do We Need Blood Substitutes?• Provides disease-free alternative to transfusions• Universal Compatibility• Longer shelf-life than allogeneic blood• Current shortage in donors/eligible donors• Religious/Social implications• Military/Trauma applications• Other applications requiring oxygen delivery

(such as organ delivery)• Less costly

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What Is In Blood?

• Plasma– Proteins– Water– Other solutes

• Formed Elements– White Blood Cells– Platelets– Red Blood Cells

Stock Photo © DennisKunkel.com

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Red Blood Cells

• Hematocrit = % of total blood volume occupied by RBCs (determined by centrifuge)

• Glycolipids in membrane account for ABO & +/-groups

• Mature RBCs lack organelles/nucleus • Anaerobic (due to lack of mitochondria) therefore

does not use up O2• Primarily contains hemoglobin (Hb)

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Physical Properties of Blood• Viscosity, non-Newtonian, dependant on

hematocrit (Hct), Temperature (T), shear rate (γ) –μ(γ,Hct, T) = (eλ/γ + ηHct)[α + φ/(1+eβ(Τ-ε))]

• Blood Flow : Q = ∆Pr4/8μl • Density• Oxygen dissociation• Non-Newtonian:

– τ = K(δu/δy)n• Pseudo-plastic or shear-thinning, thus n

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Heart Beat• Blood Pressure = Cardiac Output * Total Peripheral

Resistance• Sensory tissues on the surface of blood vessels.• Example: higher viscosity in blood would force the

pressure to go up, then the heart would work harder.• CO = heart rate * stroke volume• TPR = 8µl/r4pi• Vasoconstriction

Hypertension.Nitric oxide help dilate vessels

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Pulsatile Flow and Blood Pressure

Reference (13)

Reference (10)

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Arteries

• Elastic arteries are the largest arteries and receive the main output of the left ventricle

• The elastic tissue in their walls provides the resilience to smooth out the pressure wave

• Muscular arteries have a media composed almost entirely of smooth muscle

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Arteriolar Vessels

• Small vessel radius compared to arteries• High resistance area results in large

pressure drops, smoothing out the pulsatileblood flow from the heart

• Large amounts of smooth muscles compared to tube radius

• Arteriovenous shunts bypass capilliaries

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Capillaries• Major site of matter exchange• Microvasculature • Tiny radius on the scale of µm• Short distance to tissue• Slow & steady blood flow• Large surface area (~700 times that of aorta)• Drains into a series of vessels of increasing

diameter to form venules and veins • Oedema occurs when pressure is high

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Non-Newtonian Blood

• Blood flow in capillaries smaller than ~1mm• Viscosity depends on vessel radius• Plasma Skimming:

Decrease in blood vessel size induces decrease in blood viscosity due to the formation of cell free zone near cell wallCell free zone between 1µm -5µm in blood with a hematocrit between 40% ~ 50%

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Cell Deformation

• Red blood cell have a biconcave shape and an average diameter of about 8 µm. To pass through the very narrow capillaries they undergo elastic deformation.

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Hemoglobin

• Most blood substitutes refer to Hb substitutes derived from RBCs

• Hb in the RBC is composed of two pairs of protein sub-units which merge to produce a tetramer

β

α β

α

-Hb undergoes a conformational change when it releases O2 and picks up CO2

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Oxyhemoglobin Dissociation Curve

• P50 is the partial pressure of oxygen in the blood at which the hemoglobin is 50% saturated, typically about 26.6 mmHg Right shift: lower affinity for O2

Left shift: larger affinity for O2

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Shift Factors

• Increase in CO2 just reacts with water to get H+CO2+ H2O H+ + HCO-3

Increase DecreaseP(CO2) Shifts right Shifts rightpH Shifts left Shifts rightTemperature Shifts right Shifts leftCO Shifts right Shifts left2,3-DPG Shifts right Shifts left

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Perfluorocarbonshttp://www.allp.com/Oxygent/Graphics/oxygent9.htm• Chemically inert, hydrophobic• Currently, Oxygent is the most advanced PFC• Alliance claims it provides immediate O2 delivery• Phase 3 general surgery study in Europe: “patients

who received Oxygent either completely avoided the need for blood more frequently than the control group, or required fewer units of blood than the controls.”(3) Found avoidance and reduction of blood use in 3 weeks of post-op

• Intravenous – oxygenates in lungs, delivers O2, picks up CO2, expirates in lung• Diffusive – absorbs/releases through simple diffusion, unlike Hb• Small – 0.2μ unlike RBCs 7.0μ• No binding of NO, CO (no vasoconstriction)•Adjustable viscosity• 2 year shelf life, several days half-life

Lecithin (phospholipid) (Image © Alliance Pharmaceutical Corp.)

http://www.allp.com/Oxygent/Graphics/oxygent9.htm

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Perfluorocarbons

Reference (4)

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Extraction Of Hb From RBCs• Centrifuge blood• Remove WBCs/platelets and plasma by

aspiration• Wash RBCs• Centrifuge again• Lower layer = Hb• Extract stroma-lipid and cellular debris

through aspiration• Dialyze Hb in Ringer’s Lactate Solution

Plasma & Solutes- yellowish

WBCs/Platelets- “buffy” layer

RBCs-deep crimson

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The Beginnings of Hb Substitutes

• Lysed RBC membrane stroma is toxic to kidney• Animal testing of “stroma-free” Hb is effective

and successful– Human testing produced renal and cardiovascular

problems

• Found that Hb is toxic outside of the RBCs, breaks into 2 dimers which cannot be metabolized by the kidneys

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Initial Modifications

See Reference (1)

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Clinical Trial Phases for Major Types

Table was compiled in July of 2004 (5)

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Encapsulated and X-linked Hb

• First Generation Blood Substitute• Solved dimer problem by intramolecular

crosslinkage of dimers and by artificial phospholipid bilayer keeping Hb inside a vesicle

• Small size meant able to bypass blockages to reach smaller vessels

• High P50 meant easy release of O2

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Issues with 1st Generation• Small molecules can bypass

blockages but:– Thins blood, reducing shear

stress in capillaries and resulting in vasoconstriction

– Molecules are now able to diffuse through blood vessel, mopping up NO

• Low oxygen affinity means O2 is easily diffused but:– O2 is too easily let go, diffuses

in arteries instead of capillary beds!

Vascular W

all

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Polymerized Hb• Creates chains of hemoglobin

– Get larger molecules that won’t mop up NO– Increases shear stress

• Still have low oxygen affinity– Gives up oxygen more easily

• Two major brands in clinical phase III• PolyHeme (Northfield Labs, over 1 year shelf life)

– Clinical Trials in Trauma being conducted through exception in informed consent requirements in the U.S. (21 CFR. 50.24) (Ethical Implications)

• Hemopure (Biopure)– Study in 2000 found adverse effects, have since been

rectified and are continuing in clinical testing

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Conjugated Hb• MP4 (Hemospan by Sangart)• Six strands of PEG are attached to

HbLarger molecular radius

– Higher viscosity– Lower diffusion coefficient– More similar to blood

• Lower P50– Won’t let go of O2 until capillary

beds, sends O2 to most deprived tissue

• Phase II trial found no adverse affects (including those normally associated with blood substitutes)

Reference (9)

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Conjugated Vs Polymerized

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Blood Pressure Effects

• Hypertension occurs after 30 minutes in both crosslinked and polymerized forms (8)

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Oxygen Dissociation• O2 release in

encapsulated about equal in both

• O2 release in poly is greater in arterioles

• O2 release in MP4 is greater in capillary beds (8)

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Recombinant Hb(3rd Generation)

• New method of producing hemoglobin• Using e.coli bacteria to proliferate hemoglobin• No use of human or bovine blood components

at all• Potentially limitless supply of blood

replacement for trauma and surgery• Genetic Engineering

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References1) Chang, Thomas M.S. “Artificial Cells & Organs Research Centre” McGill University

http://www.medicine.mcgill.ca/artcell/ Retrieved Sept 26, 2005.2) Chang, Thomas M.S.. “Blood Substitutes: Principles, Methods, Products and Clinical

Trials” Tissue Engineering: Volume I. Karger Landes Systems. Montreal, QC. ©1997

3) “Oxygent,” Alliance Pharmaceutical Corp. http://www.allp.com/Oxygent/OX.HTMRetrieved Oct 30, 2005.

4) Riess, Jean G. “Understanding the Fundamentals of Perfluorocarbons and Perfluorocarbon Emulsions Relevant to In Vivo Oxygen Delivery” Artificial Cells, Blood Substitutes, and Biotechnology, 33: 47–63, 2005

5) “The Search for a Blood Substitute,” United Blood Services: Blood News. July 2004 Edition

6) Wang, Linda. “Blood Relatives” Science News. March 31, 2001 Volume 159, p206. Retrieved Sept 29, 2005 on InfoTrac

7) Westphal, S.P. “Breaking the Rules on Artificial Blood.”, The New Scientist. March 13, 2004 Volume 181, p8. Retrieved Sept 29, 2005 on InfoTrac

8) Winslow, Robert M. “Targeted O2 Delivery by Low-p50 Hemoglobin: A New Basis for Hemoglobin-based Oxygen Carriers”, Artificial Cells, Blood Substitutes, and Biotechnology, 33: 1–12, 2005

9) Björkholm, M, Fagrell, B. et al. “A phase I single blind clinical trial of a new oxygen transport agent (MP4), human hemoglobin modified with maleimide-activated polyethylene glycol” Haematologica. Issue 90, Volume 4, 2005.

10) “Blood Pressure,” John Ross. http://greenfield.fortunecity.com/rattler/46/vessels.htm. Retrieved Oct 30, 2005.

11) “Gas Exchange”, Blood Gases Manual. http://www.madsci.com/manu/indexgas.htm. Retrieved Oct 30, 2005.

12) “The Search for a Blood Substitute,” United Blood Services: Blood News. July 2004 Edition.

13) S. A. Berger, E. W. Goldsmith, and E. R. Lewis, "Introduction to Bioengineering," Oxford University Press, 2000

Blood SubstitutesWhy Do We Need Blood Substitutes?What Is In Blood?Red Blood CellsPhysical Properties of BloodHeart BeatPulsatile Flow and Blood PressureArteriesArteriolar VesselsCapillariesNon-Newtonian BloodCell DeformationHemoglobinOxyhemoglobin Dissociation CurveShift FactorsPerfluorocarbonsPerfluorocarbonsExtraction Of Hb From RBCsThe Beginnings of Hb SubstitutesInitial ModificationsClinical Trial Phases for Major TypesEncapsulated and X-linked HbIssues with 1st GenerationPolymerized HbConjugated HbConjugated Vs PolymerizedBlood Pressure EffectsOxygen DissociationRecombinant Hb�(3rd Generation)References