P. ZACHEEMD PhD

ALGEMEENZIEKENHUIS

STUIVENBERG

UNIVERSITAIREZIEKENHUIZENLEUVEN

HEM/91000P

William Harvey (1578-1657)Exercitatio Anatomica de Motu Cordis

et Sanguinis in Animalibus"... Door een bloedvat af te binden, kunnen we aantonen dat het bloed van de slagaders naar de aders stroomt, reden om aan te nemen dat het bloed continu circuleert met het hart als aandrijfkracht.De vraag is nu of dit is bedoeld voor de voeding ofvoor het behoud van bloed en longen door warmte tetransporteren , zodat het in armen en benen afgekoelde bloed in het hart opnieuw wordt opgewarmd. ..."

Transport and communication system

Host DefenceCoagulationOncotic pressureNutrient & wasteThermoregulationOxygen carrier

Cell membrane

O2

2,3 DPG

a1

a2 b

2

b1

molecule

(tetramer)

O2

2,3 DPG

a1

a2 b2

b1

Hemoglobin molecule(tetramer)

HEM/90213P

Bloodtransfusion

�Goal:� increase in D02 and VO2

�Strategy:� fluid ---> to optimalise the fillingpressure� RBC-mass ---> to increase the DO2

Pulmonary factorsdiffusion

Pa02 VentilationPerfusion

FiO2 (altitude)

HB concentration

Sa02

(=220-age)Sinus node dysfunctionDrugs ( B-blockers)

Genetic factorsHeart size

Conditioning factorsContractilityAfter LoadPreload

Disease factorsWall motionVentricular functionValve stenosis or regurgitation

500 ml/min/m2

Influence of packed cell volume (PCV) on the relative oxigen transport capacity of blood

110

90

70

50

30

10

200

150

100

50

0

20.0

15.0

10.0

5.0

10 30 50 70

Relative oxigen transport capacity

Viscosity

Hemoglobin

Hem

oglo

bin

(%)

Visc

osity

(cP)

PCV

Rel

ativ

eox

igen

tran

spor

t cap

acity

(%)

HEM/90221P

Pulmonary factorsdiffusion

Pa02 VentilationPerfusion

FiO2 (altitude)

HB concentration

Sa02

(=220-age)Sinus node dysfunctionDrugs ( B-blockers)

Genetic factorsHeart size

Conditioning factorsContractilityAfter LoadPreload

Disease factorsWall motionVentricular functionValve stenosis or regurgitation

Skeletal musclesAerobic enzymesFiber typeMuscle disease

Capillary density

Cardiogenic shock Septic shock

800600400200

015010050

06420

DO2ml/min/M2

VO2ml/min/M2

lactatemEq/L

Pretransfusion Posttransfusion

HEM/90222P

Why increase in DO2 and not in VO2?

• Low 2,3-DPG in packed cell• reduction in RBC deformability• Increase in blood viscosity• dysfunction in the microcirculation• Heterogenicity in vascular conductivity

• High flow (vasodillatation) increase in DO2 when high HT• Low flow (vasoconstriction) increase in DO2 when low HT

BLOOD substitution Clinical situation

• Preoperative status• Chronic anaemia• Acute anaemia

• Bleeding• Acute operation• Sepsis ICU patiënt

Jehovah’s Witnesses

� Agree with EPO, IV Fe, folinic acid, PFC, human HBOC, recombinant HBOC, PFC

� Not agree with transfusion allo or auto, mammalian HBOC

� General rule: avoid risk situations for your patients

Preoperative status

• Cardiopulmonary evalutation• Correction of anaemia before operation• Correction of coagulation disorders: anamnesis, PTT,

APTT, fibrinogen, PFA, platelet count• If large bloodloss is expected: 4 U PC

• Pre-op IV EPO 300-500U/Kg IV D-3 to D+2• folinic acid 15 mg IV /d D-3 to D+5• IV iron 200 mg D-3 to D0

Hem

atoc

rit(1

/1)

Tfr/f

tn

1500

1000

500

0

0 5 10 15 20 25Time (days)

0.46

0.44

0.42

placebo (n=10)rHuEpo (n=19)

Gareau et al, 1996 HEM/70675M

45

35

25

150 4 8 12 16

Weeks of rHuEpo Therapy

Hem

atoc

rit(%

)500 U/kg

150 U/kg

50 U/kg

15 U/kg

Brown et al, 1993 ACZA 2 PZ

Pharmacokinetics of EPO

300 E/Kg SC op D1EPO in serum (mE/ml) 300 E/Kg IV op D1

10000

1000

100

108 24 36 48185 15 302 12

Tijd (uren)

FLUORESENCE INTENSITY FLUORESENCE INTENSITY

BEFORE: rH-EPO AFTER: rH-EPOfr

eque

ncy

f req

uenc

y

Effect of EPO on reticulocyte age in CHD patients

RBC enzymes during EPO therapy

10067.637.588 +/- 70Retic X109/L

16.9514.6710.7210.6-16.2DPG umol/g Hb

3.363.53.61.97-3.29ATP umol/gHb

7.195.955.654,10-7.90G6PD U/gHb

9.849.097.893.80-12.0PK U/gHb

1.711.521.090.64-0.90Hx U/gHb

During EPOAfter 1 mthBefore EPONl Limits

Chronic anaemia

�Hematological consult + adapted treatment�Hematological growth factors, substitution of

deficiencies�Pharmacological agents for prophylaxis and treatment

of bleeding (e.g tranexamic acid, epsilon-aminocaproic acid, aprotinine, desmopressin)

�Restricted phlebotomy for laboratory tests�Hormone manipulation to control menstrual blood loss�Supplemental oxygen to alleviate symptoms of anaemia�(Oxygen-carrying red-cell substitutes) no effect

Pulmonary factorsdiffusion

Pa02 VentilationPerfusion

FiO2 (altitude)

HB concentration+ P50

Sa02

(=220-age)Sinus node dysfunctionDrugs ( B-blockers)

Genetic factorsHeart size

Conditioning factorsContractilityAfter LoadPreload

Disease factorsWall motionVentricular functionValve stenosis or regurgitation

Skeletal musclesAerobic enzymesFiber typeMuscle disease

Capillary density

Acute Anaemia

• Maintain Q, peroperativ hemodilution• Reduction of transfusion trigger

• except: cardio&pulmonary disease, old age, betablockers• Normovolemic hemodilution

• Pre-op 750-1500 ml, compensation with colloÏd:cristaloid• decrease in bloodviscosity, increase of Q, increase of coronary

flow, increase in O2 extraction• When 20% loss of RCM is expected• Except: cardio&pulmonary disease, old age, betablockers, renal

disease, coagulation disorders, fever, pain, or when VO2 is increased.

Acute Anaemia• Blood cell salvage per and post operatively

• Except in the presence of infection• Reduction in bleeding

• Anesthesical and surgical techniques: controled hypotension, loco-regional, endoscopic, operation field on the top, disection with laser or electro-cauter, bone waks.

• Pharmacological agents for prophylaxis and treatment of bleeding

• Resticted phlebotomy for laboratory tests• Hormone manipulation to control menstrual blood loss

Acute Anaemia

• Supplemental oxygen to alleviate symptoms of anaemia• IV EPO 300-500U/Kg IV • folinic acid 15 mg IV /d• IV iron if needed (cave infection)

• Oxygen cariers

• Can be used immediately• Can be stored in a ready-for-use• formulation for years• Without transfusion reactions (non-antigenic, non-

toxic, non-pyrogenic, virus free)• The same O2 carrying capacity and delivery to the

tissues as RBC's• Adequate colloid oncotic pressure• Adequate survival in the circulation

HEM/90846P

�

�

HEM/90851P

O2

2,3 DPG

a 1

a2 b2

b1

HAEMOGLOBIN

0 20 40 60 80 100 120

Partial pressure of oxygen (mmHg)

0

20

40

60

80

100

Hb

Sat u

ratio

n (%

)

2,3 DPG 2,3 DPG

pH pH

arterialvenous

-COOPERATIVITY (heme/heme interaction)- 16 mmHg

-2,3 DPG-BOHR effect :

-CO2-Cl--PH

-HILL number (log function of the slope at P50)-2,7-3,1

-TEMPERATURE

������

��� ���

O2

2,3 DPG

a 1

a2 b2

b1

HAEMOGLOBIN

O O +H Hb HbO + H +HbO H Hb+O O

2CO 2CO2CO

H O2

3H CO2

22 22

+

+2 2

ERYTHROCYTETISSUES LUNGS

CA

+

Cl - Cl-3HCO-

3HCO-3HCO-

DPG

��� ���

��� ���DPG

��� ���

��� ���2

���

���

���

���

���

���

���

���

DPG

��� ���

������

2

22

NL= 2,7 – 3,1

1= high affinity Hb

OXY-Hb --> DEOXY-HbP50 > 30 mmHg

-COOPERATIVITY <16 mmHg

-2,3 DPG-BOHR effect

-Low PH-High CL-

-High C02-HILL number (log function of the slope at P50)

>2,7-3,1-TEMPERATURE

-High temperature

High-affinity, noncooperative, cell-free hemogobin

P50

H+CO22,3-DPGCl-temperature

0 20 40 60 80 100 120 140 PO2 (Torr)

Saturation (fraction) Normal blood

0.8

0.6

0.4

0.2

0.0

ACZA 1PZ

O2

a1 b1• Low P50 (no 2,3-DPG)• Tetramer to Dimers• Low half-life (1-2hrs)• Iso-oncotic at 7 g/dl• Renal toxicity• Vasoconstriction

a2 b2

a1b1

a2 b2

HEM/70565P

Hypertension & gastrointestinal• Stroma• NO

Ca2+ NO synthase activity

NO Citrulline L-Arginine

Endothelial cell

Guanylate cyclase activity (Fe 2+ )

Smooth muscle cell

blood

GTP cGMP Ca2+ Vasodilation

Hb Fe2+ Stimuli (Ach)

HEM/90872P

• To decrease oxygen affinity

• To prolong intravascular retention

• To decrease colloid osmotic activity

• To prevent toxicity (renal & extravasation & hypertension & gastrointestinal)

HEM/90850P

bis-(3.5-dibromosalicyl) fumaratecross-link two a subunitsmodifying the 2.3-DPG pocket (increase in P50)Diaspirin cross-linked tetrameric Hb Phase III trials

O 2

a 1

a 2 b2

b1

O 2

O 2 O 2

HEM/90857P

• engineered in E-coli• prevent breakdown of the tetramers• high P50

b2

b1a1

a2

HEM/90858P

• glutaraldehyde cross-linking• pyridoxal phosphate increase the P50

• bovine poly Hb P50 is controlled by Cl

a1

a 2 b 2

b1

PLPPLP

a1

a 2 b 2

b1

PLPPLP

a1

a 2 b 2

b1

PLPPLP

Tetrameric Hb < 1%

HEM/90856P

O2

a1

a 2 b2

b1

O2

O2 O2

PLPPLP

POE POE

POEPOE

POE

POE

POE

POE

O 2

a 1

a 2 b2

b1

O 2

O 2 O 2

PLPPLP

POE POE

POEPOE

POE

POE POE

O 2

a 1

a 2 b 2

b1

O 2

O 2 O 2

PLPPLP

POE POE

POEPOE

POE

POE

• dextran• polyoxyethylene• polyethylene glycol

HEM/90859P

� Limiting binding of NO by extravascular Hb� Half-life reduced due to removing by spleen and liver� Interaction with granulocytes and immune system

2,3 DPG

a1

a2 b2

b1

2,3 DPG

a1

a2 b2

b1

2,3 DPG

a1

a2 b2

b1

2,3 DPG

a1

a2 b2

b1

2,3 DPG

a1

a2 b2

b1

2,3 DPG

a1

a2 b2

b1

2,3 DPG

a1

a2 b2

b1

0.2µm NO

HEM/90866P

P50 of some new hemoglobins

49.5 mmHg5.6 g/dlNeo Red Cells® Terumo (human Hb)

Encapsulated Hb

ca. 15 mmHg6 g/dlPEG-Hb Enzon (bovine Hb)

Polymerized-conjugated Hb

38 mmHg13 g/dlHemopure® Biopure / Upjohn (bovine Hb)

34 mmHg10 g/dlHemolink® Hemosol (human Hb)

28-30 mmHg10 g/dlPolyHeme® Northfield (human Hb)

Polymerized Hb

32 mmHg5-8 g/dlOptro® Somatogen / Eli Lilly (rHu-Hb)

32 mmHg10 g/dlHemAssist® Baxter (human Hb)

Crosslinked Hb (intramolecular)P50Hb concentration

Hill coefficient of some new hemoglobins

n.a.5.6 g/dlNeo Red Cells® Terumo (human Hb)Encapsulated Hb

16 g/dlPEG-Hb Enzon (bovine Hb)Polymerized-conjugated Hb

n.a.13 g/dlHemopure® Biopure / Upjohn (bovine Hb)110 g/dlHemolink® Hemosol (human Hb)

n.a.10 g/dlPolyHeme® Northfield (human Hb)Polymerized Hb

2.275-8 g/dlOptro® Somatogen / Eli Lilly (rHu-Hb)n.a.10 g/dlHemAssist® Baxter (human Hb)

Crosslinked Hb (intramolecular)Hill coefficientHb concentration

HEM/00492M

COP of some new hemoglobins

25 mmHg5.6 g/dlNeo Red Cells® Terumo (human Hb)

Encapsulated Hb

ca. 100 mmHg6 g/dlPEG-Hb Enzon (bovine Hb)

Polymerized-conjugated Hb

17 mmHg13 g/dlHemopure® Biopure / Upjohn (bovine Hb)

24 mmHg10 g/dlHemolink® Hemosol (human Hb)

n.a.10 g/dlPolyHeme® Northfield (human Hb)

Polymerized Hb

16 mmHg5-8 g/dlOptro® Somatogen / Eli Lilly (rHu-Hb)

43 mmHg10 g/dlHemAssist® Baxter (human Hb)

Crosslinked Hb (intramolecular)

COPHb concentration

HEM/00493M

Circulation storage shelf life & half life

ca. 44 h1 year frozenPEG-Hb Enzon (bovine Hb)Polymerized-conjugated Hb

ca. 24 h2 years room temperature

Hemopure® Biopure / Upjohn (bovine Hb)

ca. 14 h2 years refrigeration

Hemolink® Hemosol (human Hb)

24 h12 months refrigeration

PolyHeme® Northfield (human Hb)Polymerized Hb

4-5 h18 months refrigeration

Optro® Somatogen / Eli Lilly (rHu-Hb)ca. 14 hn.a.HemAssist® Baxter (human Hb)

Crosslinked Hb (intramolecular)CirculationShelf life storage

Viscosity of some new hemoglobins

2 cP5.6 g/dlNeo Red Cells® Terumo (human Hb)Encapsulated Hb

n.a.6 g/dlPEG-Hb Enzon (bovine Hb)Polymerized-conjugated Hb

1.3 cP13 g/dlHemopure® Biopure / Upjohn (bovine Hb)1 cP10 g/dlHemolink® Hemosol (human Hb)n.a.10 g/dlPolyHeme® Northfield (human Hb)

Polymerized Hbn.a.5-8 g/dlOptro® Somatogen / Eli Lilly (rHu-Hb)

1.1 cP10 g/dlHemAssist® Baxter (human Hb)Crosslinked Hb (intramolecular)

ViscosityHb concentration

HEM/00495M

Clinical Use

Phase IIRadiosenstive, chemosensitive tumors; volume

expansion

PEG-Hb Enzon (bovine Hb)Polymerized-conjugated Hb

Phase IIIElective surgery, sickle cell crisis

Hemopure® Biopure / Upjohn (bovine Hb)

Phase IIICardiac, orthopedic surgery;

erythropoiesis

Hemolink® Hemosol (human Hb)

Phase IIITrauma, elective/emergency

surgery

PolyHeme® Northfield (human Hb)Polymerized Hb

preclinicalCardiac bypassOptro® Somatogen / Baxter (rHu-Hb)Crosslinked Hb (intramolecular)

Regulatory phase

Potential clinical use

Artificial O2 carriersThe modified hemoglobin-based solutions are grouped by the

source of the hemoglobin• Modified Hemoglobin-Based Solutions

• Derived from Outdated Human Red Cells• Polymerized hemoglobin (PolyHeme®, Northfield) (phase III clinical trials)• Polymerized and cross-linked hemoglobin (Hemolink®, Hemosol) (phase III clinical

trials) • Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP, Apex Bioscience)

(phase III clinical trials) • Diaspirin cross-linked hemoglobin (HemAssist®/DClHb®, Baxter) (discontinued)

• Derived from Bovine Red Cells• Polymerized Purified Bovine Hemoglobin (HemoPure®, Biopure) (phase III clinical

trials) • Polyethylene-Glycol Modified-Bovine Hemoglobin (PEG-Hgb®, Enzon) (phase Ib

clinical trials)• Recombinant Human Hemoglobin

• E. coli46 (Optro® /rHb1.1, Somatogen/Baxter) (discontinued) Transgenic tobacco47

(preclinical)HEM/00501M

�

�

HEM/90870P

O2

PERFLUOROCARBON40-50% O2 dissolvingcapacity for 100% pure oxygen

HEM/90211P

High gas-dissolving capacity(O and CO )Low viscosityChemical and biologic inertInsoluble in water

,

,,,

F

F

C

C

C

C

C

C

C

C

C

C

F2F2

F2

F2 F2

F2

F2

F2

F-decalin (Fluosol-DA)

C

C

C

C

C

C

C

F3

F2 F2F2 F2

F2 F2 F2

Br

F-octylbromide (oxygent)

2 2

Emulsion of 0.1-0.3 umSurface active agent (surfactant)

poloxamer:Pluronic F-68 (Fluosol-DA) (allergic reactions)egg-yolk phospholipide

T 1/2: 4 to 7 daysexhalation or phagocytosis (RES)Problem: coalescence of the emulsion

Compared solubilities of the respiratory gases in perfluorocarbons and their emulsion

15.350% Emulsion of perfluorooctylbromide (perflubron)707.520% Emulsion of F-decalin (Fluosol)

50Perfluorooctylbromide C8F17Br13045F-decalin16645.3F-tripropylamin N (C4F7)3

14240.3F-tributilamin N (C4F9)3

7020Blood652.3H2O, plasma

CO2O2

O2 and CO2 solubilities in fluorochemicals and their emulsions(ml/dL, 37°C, 1 atm)

Clark et all, 1982HEM/00483M

Comparisons between 1st and 2nd generation in perfluorocarbon emulsions

Egg yolk phospholipidsEmulsifierPluronic F-18 +++

Egg yolk phospholipids

17.2 ml/dlO2 carrying capacity5-7 ml/dl1.96Density1.96

30% vol/volConcentration10-11% vol/vol

Perfluoro-octylbromidePFCPerfluoro-

tripropylamidePerfluoro-

decalin

7

7.5 (10ml/kg)14.5 (20ml/kg)22 (30ml/kg)

< 600 nM

7 (1.8g/kg)Half-live tissues (days)65

4 (2.7g/kg)Half-life iv (hours)

< 250 nmSize of micelles

PerflubronFluosol DA

HEM/00485M

Oxygen equilibrium curves Hemoglobin attains nearly complete saturation at the

arterial oxygen partial pressure 100 mmHgBlood 14g/dl

Hemogobin 7g/dl

100 200 300 400 PO2 (Torr)

25

20

15

10

5

PFOB

Fluosol-DA 20%O2

cont

ent (

ml/d

l)

HEM/00489M

Artificial O2 carriersThe modified hemoglobin-based solutions are grouped by the

source of the hemoglobin

• Perfluorocarbon (PFC) Emulsions• Perflubron (Oxygent®, Alliance/Baxter) (phase III clinical trials) • Emulsified perfluorodichlorooctane (Oxyfluor® ,

HemaGen/PFC) ( discontinued)

• ‘First-Generation’ PFC emulsions• Fluosol® (Green Cross, Japan) (discontinued)• Perftoran (Russia)

HEM/00502M

Future• Blood poisoning: CO, CN-,etc.• Converting hypoxic cells in the center of the tumor to

aerobic metabolism, increasing susceptibility to radiation and chemotherapy

• Temporary vitreal replacement• Organ preservation• Radiology (CTscan, NMR) perfluorooctyl bromide• Assessment of tissue oxygenation• Ischaemia and microcirculatory disturbances• Liquid ventillation

HEM/90871P

Advantages and disadvantages of hemoglobin-based solutions

• Advantages• Carries and unloads O2

• Sigmoidal O2 dissociation curve• 100% FiO2 is not mandatory for maximal potency• Easy to measure

• Disadvantages• Side effects

• Vasoconstriction• Interference with colorimetric laboratory methods

HEM/00503M

Advantages and disadvantages of perfluorocarbon (PFC)

• Advantages• Carries and unloads O2• Few and mild side effects• No known organ toxicity

• Disadvantages• 100% FiO2 is mandatory for maximal efficacy• Additional colloid often necessary with potential

side effects

HEM/00504M

Albert Einstein

25 to 30 %

Limit of efficacy

• intravascular persistence only a few hours & days

• colloid osmotic activity• vasoactive• oxidation to methemoglobin• reperfusion injury

HEM/90862P

• Hemostasis: Hb binds NO � vasoconstriction and increased platelet adhesion

• Vasoactivity• execessive delivery of O2 to arterioles � increase in endothelin

� vasoconstriction • Binding of NO

• Gastrointestinal side effects (NO)• Immunosuppression• Interference with laboratory assays

HEM/90861P

PFC side effects

• Flu-like symptoms

• Sequestration in spleen and liver• Hepato & splenomegaly• thrombocytopenia