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RBC storage lesions:What they are, and how we can

minimize them

Tatsuro Yoshida PhD

New Health Sciences Inc. Bethesda MD

Biomedical Engineering, Boston University

Boston MA



Storage lesions What are they?

How are they related?

What are the consequences?

What can we do about them? Prevention

Reversal Targets for ideal refrigerated RBC

storage.

Recent review literatures [1-3]



Refrigerated RBC storage:

altered parameters

Biochemical

MetabolicBiomechanical Oxidative

SNO-Hb




altered parameters

Biochemical


SNO-Hb



Storage lesions of metabolic origin

Biochemical


SNO-Hb



Storage lesions of metabolic origin

References [4,5]



Consequences of ATP depletion

References [4,5]



Consequences of ATP depletion (2)

References [4,5]



Storage lesion: loss of

vasoregulation Hemolysis [gradual increase]

NO scavenger--vasoconstriction

ATP release [gradual decrease]

Loss of ATP mediated vasodilation

Loss of NO delivery by RBC [fast]

Impaired vasoregulation



Regulation of microvascular perfusion

mediated by ATP

References [4,5] From: Ellsworth ML et al., Am J Physiol

1995;269(6 Pt 2):H2155-61.



Consequence of 2,3-DPG depletion

References [4,5]

Release DPG from Hb

Reduced oxygen delivery capacity



Consequences of depleted 2,3-DPG

²reduction in tissue oxygen delivery

From Bunn & Forget 1986 [7]



2,3-DPG and hemoglobin

References [4,5]

Release DPG from Hb

Reduced oxygen delivery capacity

Modified figure from Perutz, Nature 228:734,1970



Loss of NO-linked vasoregulation by RBC

Studies: Stored blood lacks nitric oxide: Associated Press

October 8, 2007 08:06:20 PM PST

Vasodiatory activity of RBC is lost 3 hrs

after blood collection

Bennett-Guerrero E, Veldman TH, Doctor A, et al. Evolution of adverse changes in stored RBCs. Proceedings of the National Academy of Sciences 2007;104(43):17063-68.



RBC is a carrier of NO for vasoregulation

References [27,28]

Regulation of microvascular perfusion

mediated by Hemoglobin + NO

SNO---biologically active S-nirtrosothiol



Hemoglobin and nitric oxide

(Hb)3Hb-NO

(HbO2

)4

-SNO

O2RSNO

O2

NO

(Hb)4 T-State

R-State

LUNG

(Hb)4-SNO



Loss of NO-linked RBC

mediated vasoregulation Immediately after transfusion, RBCs

may not only fail to increase peripheral

blood flow, but may also function as NO

sink



Loss of NO-linked RBC

mediated vasoregulation Immediately after transfusion, RBCs

may not only fail to increase peripheral

blood flow, but may also function as NO

sink

How serious is this effect in relation toother storage lesions?

Is it reversible in vivo---most likely

How fast can it recover invivo

??



Storage lesions linked to oxidative

damage

Biochemical


SNO-Hb



Oxidative damage pathway

References [8-11]



Storage lesions linked to oxidative damage

References [8-11]

SNO-Hb



Consequences: oxidative damage

References [12,13]



Damage pathwaysBiochemical

/ Metabolic

alterations

Bio-mechanical

changes

Hemolysis

Post-transfusion removal

TRALI

Oxidative

Damage



Reduction or delaying

development of storage lesions

Metabolically linked



RBC metabolic pathways

[26]



Prevention of metabolic storage lesions:

Embden-Meyerhof pathway² ATP and 23DPG

Metabolic modulation [14-15]




manipulations with additive solution

Review: Hess 2006 [16]




storage under anaerobic condition

References [17-19]



Reduction or delaying

development of storage lesions

Oxidative damages



Reduction of oxidative damage:

RBC redox system




metabolic manipulations

�GSH precursors [20]

�L-carnitine (protection of RBC

membrane [21])

�Anti-oxidant / free radical

scavenger in storage solution

Mannitol, N-acetylcysteine

tirilazad mesylate, etc

[11,22,23]




storage under anaerobic condition

Store RBC without oxygen� Stop hydroxyl radical-

mediated peroxidation cycles

� Prevent hemoglobindenaturation

Reference [19]



Reversal of storage lesions



Reversibility of storage lesions

SNO-Hb

?



Rejuvenation Post-storage metabolic manipulations

Mixture of pyruvate, inosine, Pi,

adenine, PEP etc

Rejuvesol (Cytosol Laboratory Inc)

37rC incubation followed by cell washing [24]

Experimental PEP [references in 25]



RBC metabolic pathways

[26]



Rejuvenation by PIPA (Rejuvesol)



Rejuvenation by PIPARejuvenation by PIPA (Rejuvesol)



Rejuvenation with PEP

Reference [25]



Result of rejuvenation Restored ATP

Elevated 2,3-DPG

Increased 24-hr recovery rate Rejuvesol is approved by FDA, but RBC must be

washed



Result of rejuvenation Restored ATP

Elevated 2,3-DPG

Increased 24-hr recovery rate Rejuvesol is approved by FDA, but RBC must be

washed

Rejuvesol can be added during storage High levels of ATP and 2,3DPG throughout

extended storage period

Prevent PS accumulation on RBC surface

Increased 24-hr recovery rate



Goals for improved RBC

storage



Reduced toxicity Reduce hemolysis

Reduce dead-on-arrival RBC Iron toxicity

Vasoconstriction via NO scavenging

Maintain deformability / prevent aggregation Prevent capillary blockage



Increase 24-hr recovery rate and / or

extend shelf-life

Maintenance of high ATP

Minimize oxidative damage

Maintain below threshold parameters causing

removal after transfusion [to provide sufficient

time for recovery in body after transfusion]



Targets for ideal refrigerated RBC storage

Reduced toxicity Reduce hemolysis

Reduce dead-on-arrival RBC Iron toxicity

Vasoconstriction via NO scavenging

Maintain deformability / prevent aggregation Prevent capillary blockage

Prevent release, and/or remove bio-activesubstances Prevention of TRALI

Leukoreduction, irradiation, pathogen-reduction, etc.



Higher functionality for efficient

tissue oxygenation

Maintain high levels of:

2,3-DPG[oxygen release]

SNO-Hb [regulation of vascular perfusion]

ATP [regulation of vascular

perfusion]

Deformability [capillary perfusion]



6HOHFWHGUHFHQWUHYLHZVStorage lesion

Ho J, Sibbald WJ, Chin-Yee IH. Effects of storage on efficacy of red cell transfusion: when is itnot safe? Crit Care Med 2003;31(12 Suppl):S687-97.

Chin-Yee I, Arya N, d'Almeida MS. The red cell storage lesion and its implication for transfusion. Transfus Sci 1997;18(3):447-58.

Clinical consequences of transfusion (not covered in this talk)

Tinmouth A, Fergusson D, Yee IC, Hebert PC. Clinical consequences of red cell storage in thecritically ill. Transfusion 2006;46(11):2014-27.

Solheim BG, Flesland O, Seghatchian J, Brosstad F. Clinical implications of red blood cell andplatelet storage lesions: an overview. Transfus Apher Sci 2004;31(3):185-9.

Vincent JL, Baron JF, Reinhart K, et al. Anemia and blood transfusion in critically ill patients.Jama 2002;288(12):1499-507.

Spiess BD. Blood transfusion: the silent epidemic. Ann Thorac Surg 2001;72(5):S1832-7.

Additive solutions

Hess JR. An update on solutions for red cell storage. Vox Sang 2006;91(1):13-9.

Vasoregulatory function of stored RBC Bennett-Guerrero E, Veldman TH, Doctor A, et al. Evolution of adverse changes in stored.

RBCs. Proceedings of the National Academy of Sciences 2007;104(43):17063-68.



5HIHUHQFHV

1. Tinmouth A, Fergusson D, Yee IC, Hebert PC. Clinical consequences of red cell storage in the critically ill.Transfusion 2006;46(11):2014-27.

2. Ho J, Sibbald WJ, Chin-Yee IH. Effects of storage on efficacy of red cell transfusion: when is it not safe?Crit Care Med 2003;31(12 Suppl):S687-97.

3. Chin-Yee I, Arya N, d'Almeida MS. The red cell storage lesion and its implication for transfusion. Transfus

Sci 1997;18(3):447-58.4. Ellsworth ML, Forrester T, Ellis CG, Dietrich HH. The erythrocyte as a regulator of vascular tone. Am J

Physiol 1995;269(6 Pt 2):H2155-61.

5. Sprague RS, Ellsworth ML, Stephenson AH, Lonigro AJ. ATP: the red blood cell link to NO and localcontrol of the pulmonary circulation. Am J Physiol 1996;271(6 Pt 2):H2717-22.

6. Burnstock G, Kennedy C. A dual function for adenosine 5'-triphosphate in the regulation of vascular tone.Excitatory cotransmitter with noradrenaline from perivascular nerves and locally released inhibitoryintravascular agent. Circ Res 1986;58(3):319-30.

7. Bunn H, Foget B. Hemoglobin: Molecular genetics and clinical aspects. Philadelphia, PA: WB Sanders,1986.

8. Wolfe LC, Byrne AM, Lux SE. Molecular defect in the membrane skeleton of blood bank-stored red cells.Abnormal spectrin-protein 4.1-actin complex formation. J Clin Invest 1986;78(6):1681-6.

9. Wolfe LC. Oxidative injuries to the red cell membrane during conventional blood preservation. SeminHematol 1989;26(4):307-12.

10.Beppu M, Mizukami A, Nagoya M, Kikugawa K. Binding of anti-band 3 autoantibody to oxidativelydamaged erythrocytes. Formation of senescent antigen on erythrocyte surface by an oxidative mechanism. J

Biol Chem 1990;265(6):3226-33.



5HIHUHQFHV

11. Knight JA, Searles DA. The effects of various antioxidants on lipid peroxidation in storedwhole blood. Ann Clin Lab Sci 1994;24(4):294-301.

12. Silliman CC, Voelkel NF, Allard JD, et al. Plasma and lipids from stored packed red bloodcells cause acute lung injury in an animal model. J Clin Invest 1998;101(7):1458-67.

13. Boas FE, Forman L, Beutler E. Phosphatidylserine exposure and red cell viability in red cell

aging and in hemolytic anemia. Proc Natl Acad Sci U S A 1998;95(6):3077-81.

14. Messana I, Ferroni L, Misiti F, et al. Blood bank conditions and RBCs: the progressive lossof metabolic modulation. Transfusion 2000;40(3):353-60.

15. Messana I, Orlando M, Cassiano L, et al. Human erythrocyte metabolism is modulated by theO2-linked transition of hemoglobin. FEBS Lett 1996;390(1):25-8.

16. Hess JR. An update on solutions for red cell storage. Vox Sang 2006;91(1):13-9.

17. Asakura T, Sato Y, Minakami S, Yoshikawa H. Effect of deoxygenation of intracellular hemoglobin on red cell glycolysis. J Biochem (Tokyo) 1966;59(5):524-6.

18. Hamasaki N, Rose ZB. The binding of phosphorylated red cell metabolites to humanhemoglobin A. J Biol Chem 1974;249(24):7896-901.

19. Yoshida T, Aubuchon JP, Tryzelaar L, et al. Extended storage of red blood cells under anaerobic conditions. Vox Sang 2007;92(1):22-31.

20. Dumaswala UJ, Zhuo L, Mahajan S, et al. Glutathione protects chemokine-scavenging andantioxidative defense functions in human RBCs. Am J Physiol Cell Physiol2001;280(4):C867-73.



5HIHUHQFHV

21. Arduini A, Holme S, Sweeney JD, et al. Addition of L-carnitine to additive solution-suspended red cells stored at 4 degrees C reduces in vitro hemolysis and improves in vivoviability. Transfusion 1997;37(2):166-74.

22. Epps DE, Knechtel TJ, Bacznskyj O, et al. Tirilazad mesylate protects stored erythrocytesagainst osmotic fragility. Chem Phys Lipids 1994;74(2):163-74.

23. Racek J, Herynkova R, Holecek V, et al. Influence of antioxidants on the quality of stored blood. Vox Sang 1997;72(1):16-9.

24. Valeri CR. Use of rejuvenation solutions in blood preservation. Crit Rev Clin Lab Sci1982;17(4):299-374.

25. Hamasaki N, Yamamoto M. Red blood cell function and blood storage. Vox Sang2000;79(4):191-7.

26. Tanaka KR, Zerez CR. Red cell enzymopathies of the glycolytic pathway. Semin Hematol

1990;27(2):165-85.

27. Reynolds JD, Ahearn GS, Angelo M, et al. S-nitrosohemoglobin deficiency: Amechanism for loss of physiological activity in banked blood. Proceedings of

the National Academy of Sciences 2007: ;104(43):17058-62.

28. Bennett-Guerrero E, Veldman TH, Doctor A, et al. Evolution of adverse changes in storedRBCs. Proceedings of the National Academy of Sciences 2007;104(43):17063-68.

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