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Frozen red cells for military and civil purposes
Relevance, experiences and developmentsLelkens, C.C.M.
Publication date 2017
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Lelkens, C. C. M. (2017). Frozen red cells for military and civil purposes: Relevance, experiences and developments.
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Frozen Red Cells for Military and Civil Purposes
C
.C
.M.
Lelk
ens
C.C.M. Lelkens
Frozen Red Cells
for Military and Civil Purposes
Twee erythrocyten uit Leiden Besloten de warmte te mijden.
Naar de MBB, Zo spraken de twee,
Colofon
ISBN (book): 978-90-826959-0-8
ISBN (digital document): 978-90-826959-1-5 Author: Charles Chrétien Marie Lelkens Cover design: Evelien Jagtman, Maastricht Print: Drukkerij Mostert en Van Onderen, Leiden Copyright © 2017 Charles Lelkens
Alle rechten voorbehouden. Niets uit deze uitgave mag worden verveelvoudigd, opgeslagen in een geautomatiseerd gegevensbestand of openbaar gemaakt worden in enige vorm of op enige wijze, hetzij elektronisch, mechanisch of door fotokopieën, opname, of op enige andere manier, zonder voorafgaande schriftelijke toestemming van de auteur.
All rights reserved. No part of this publication may be reproduced, stored in retrieval systems, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the author.
Deze uitgave kwam tot stand dankzij een genereus gebaar van de schrijver.
Frozen Red Cells for Military and Civil Purposes
Relevance, Experiences and Developments
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus
prof. dr. ir. K.I.J. Maex
ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Aula der Universiteit
op vrijdag 30 juni 2017, te 11.00 uur door
Charles Chrétien Marie Lelkens geboren te Leiden
Promotiecommissie
Promotor: Prof. dr. A. J. Verhoeven AMC-UvA
Copromotores: Dr. J.W.M. Lagerberg Sanquin Research
Dr. D. de Korte Sanquin Research
Overige leden: Prof. dr. E. van der Schoot AMC-UvA
Prof. dr.ir. C. Ince AMC-UvA
Prof. dr. T.M. van Gulik AMC-UvA
Prof. dr. J.R. Hess University of Washington
Dr. G.J.C.G.M. Bosman Radboudumc
Dr. H. Woelders Wageningen University & Research
Faculteit der Geneeskunde
Aan mijn ouders, in liefdevolle en dankbare herinnering Aan Jacqueline, Marie-Christine en Jean-Louis
“In God we trust, from others we need data”
Carlo Robert Valeri, MD (1932) Director Naval Blood Research Laboratory,
Contents
Chapter 1 Introduction
Chapter 2 Stability after thawing of RBCs frozen with the high- and low-glycerol method. (Transfusion. 2003; 43(2):157-64) Chapter 3 Experiences with frozen blood products in the Netherlands
military. (Transfus Apher Sci. 2006; 34(3):289-98)
Chapter 4 Australian experience with frozen blood products on military operations. (Med J Aust. 2010; 192(4):2035)
Chapter 5 Prolonged postthaw shelf life of red cells frozen without prefreeze removal of excess glycerol. (Vox Sang. 2015; 108(3):219-25)
Chapter 6 The effect of prefreeze rejuvenation on postthaw storage of red cells in AS-3 and SAGM. (Accepted by Transfusion)
Chapter 7 Advances in military, field, and austere transfusion medicine in the last decade. (Transfus Apher Sci. 2013; 49(3):380-6)
Chapter 8 General discussion
Chapter 9 Summary
Chapter 10 Samenvatting
Appendices Acknowledgments / Dankwoord Curriculum vitae auctoris Resume 11 25 45 67 79 95 119 137 153 159 165 169 171
Chapter
Introduction
1
“Mijn” schip: standaardfregat Hr.Ms. Banckert (1980-1981) bron: Nederlands Instituut voor Militaire Historie, beeldbank.
Introduction
13
Introduction
Large-scale military conflicts (or “wars”) have undeniably played an important role in the progress of medical care with, for instance, the ligature, the plaster cast and the wheel stretcher as obvious examples. Civilian medical practice today still benefits from these war-driven innovations, introduced by military doctors with battlefield experience. The same holds true for several developments in blood transfusion, with the use of frozen (or cryopreserved) red blood cells as most outstanding example. For this reason, the history of the development of cryopreserved red blood cells for transfusion will be described in this chapter according to the time scale determined by major military conflicts.
The US Civil War
The first case reports of a successful military blood transfusion concerned two wounded soldiers in the US Civil War (1861-1865). In 1864, two Union Army surgeons, assigned to two different hospitals, wide apart, decided to transfuse two soldiers with human blood from healthy individuals after a leg amputation.1,2 Both soldiers survived.
In the 1890’s the potential of citrate as an anticoagulant was discovered, but not put into clinical practice until its independent rediscovery by three researchers in three different countries in 1914 and 1915.3,4 This paved the way for a safe preservation of blood for future use.
The First World War
Although in April 1915 the first recorded transfusion of World War I (and of the 20th century) was administered,5 it was not before 1916 that Canadian Captain Lawrence Bruce Robertson published the first article about his experiences with transfusing (uncrossmatched) blood via syringe and cannula in war time circumstances.6 Despite the fact that another Canadian, Major Edward Archibald, decided to introduce the use of sodium citrate in 1915,7 that allowed him to transfuse blood when needed,8Robertson preferred saline to flush syringe and cannula periodically. Like most surgeons at that time, he was very cautious in adding substances to blood. Until then, most transfusions were performed directly via end-to-end anastomosis of artery and vein of donor and recipient respectively.9 This technique proved to be totally unsuitable for emergency purposes, because it not only required two skilled surgeons and nursing staff,
Chapter 1 Introduction but also took precious time and the proximity of an available donor in the
operating room. In addition, there was always the danger of overdonation or undertransfusion.3,4,10-12
In the first year of World War I, the US Army ordered further exploration of this field, leading to experiments by Rous and Turner, who in 1916 published their findings, indicating that a mixture of 5.4% glucose and 3.8% sodium citrate could protect human red blood cells from hemolysis for four weeks.13 In April 1917, the United States entered the war and during the battle of Cambrai at the front in France in November 1917, a US military doctor, Captain Oswald H. Robertson, successfully applied the Rous-Turner solution, achieving up to 26 days of storage of whole blood.4,14 He used a self-designed icebox,14,15 containing glass bottled whole blood, thus becoming the world’s first blood banker.14 Although there are no exact figures of how many transfusions were performed during World War I, several tens of thousands in British and Canadian hospitals in 1918 appears to be a fair estimate.12
The Spanish Civil War
Almost two decades later, during the Spanish Civil War (1936-39), for the first time in history, the concept was initiated to collect and store (civilian) citrated and typed blood in a (civilian) blood bank16,17 and take it from there to the patient as close to the front as possible.18-20 The two most famous civilian blood banks were set up in Barcelona and Madrid. The transfusion service in Barcelona alone collected, processed, tested and distributed some 9,000 liters of whole blood between January 1936 and August 1939, using only group O from over 28,000 donors,16,17,21 resulting in 27,000 transfusions.
Between the two World Wars, in 1928, dr. Shamov in Ukraine suggested human cadaver blood as a safe source for transfusion. His idea was put into practice in 1930 by a Russian colleague, dr. Sergei Yudin, who in 1937 reported on having performed 1000 successful transfusions, without using citrate but relying on post mortem fibrinolysis instead.22,23 During a visit to Spain in 1934, Yudin pointed out the convenience of his idea, but it was probably never put into practice in the course of the war, at least not at the Barcelona institute.20,24 Although logical in concept, moral, ethical, but also practical consequences,22,25,26 prevented this idea from gaining solid ground in day-to-day clinical practice outside the Soviet Union, even in war time conditions, but it stimulated further development of blood conservation and blood banks.27
Between 1938 and 1940, the Russians used anticoagulated donor blood in military operations on the Kuril Islands (1938) and in Finland (1939-1940), prepared at blood transfusion institutes in Leningrad and Moscow. So when Russia entered World War II, the blood supply system was in place and, in the end, able to provide the armed forces with a total of 1.7 million liters of blood along the front lines during the war.28
The Second World War and the Korean War
The experiences in Spain and Russia in the late 1930s finally led to the deployment of blood transfusion services in World War II, capable of large-scale collections and storage of whole blood. In 1940, the work of Cohn enabled the plasma fractionation of whole blood and when the Japanese attacked Pearl Harbor, albumin was available and immediately shipped to Hawaii to treat 87 victims, mainly burn patients, of which some showed dramatic improvement.10 Although some doctors, based on their World War I experiences, had learned that severe hemorrhage led to shock and therefore had expressed their opinion that oxygen carrying capacity was the primary need, the general belief (at least in the US) was that with the availability of plasma (liquid or freeze-dried) this alone was sufficient to compensate for the blood loss. Other than that, blood transfusion was considered too difficult and dangerous and its supply was logistically much more complicated.21 Emphasis was therefore laid on the use of plasma by the US forces at the start of World War II. It was only in 1943, because of reports from the North-African theater,29 as well as the situation in Europe and in the Pacific in 1944,12that attention shifted back again to whole blood.21This persisted through the Korean War (1950-1953), during which almost unlimited amounts of whole blood were available and used to the maximum.29 Adequate resuscitation, among which the availability of blood, played an important role in reducing the numbers of wounded servicemen dying after reaching the hospital. During World War I, around 10% of those arriving at a hospital died, in World War II this number had decreased to 4.5% and finally in the Korean War to 2.6%.21
The Vietnam War
Soon after their direct military involvement in the Vietnam War (1946-1975) the US started to provide whole blood to Saigon (South Vietnam) in 1965. In that same year and the year thereafter, packed RBC and fresh frozen plasma (FFP) became largely available. Until 1971 approximately 1.3 million red
Chapter 1
16
Introduction
17
cell units were sent, of which, due to outdating, less than half (600,000, i.e. only 46%!) were administered in US hospitals. Partly because of this, the US Department of Defense (DoD) sponsored clinical research towards extension of the storage period of RBC, without yielding a licensed product though.12 Despite developments over the past decades since World War II, current preservation solutions are still not approved beyond 42 days.30 Substantial extension of (hypothermic) storage periods is possible by using cryopreservation techniques, i.e. storage below 0°C. Currently, frozen red cells can be stored for at least 10 years, according to European and US guidelines.30,31
Development of cryopreservation techniques
Commissioned by the US Navy in 1956, the Blood Research Laboratory (in 1965 renamed as the Naval Blood Research Laboratory) started to pursue another way to prolong the storage period of RBC,32 based on the findings of dr. Audrey Smith.33 She had observed that high concentrations of glycerol (45-50% w/v) could serve as a cryoprotectant for red cells, thus enabling their preservation by freezing and storing at temperatures below -65°C. In the early sixties, several investigators34-36 found that lower concentrations of glycerol (17-20% w/v), were able to act as cryoprotectant, but only if the rate of freezing was accelerated by immersion in liquid nitrogen, followed by storage below -150°C in (the vapor phase of) liquid nitrogen. These two methods, referred to as the high- (HGM) and low-glycerol method (LGM) respectively, are still in use to freeze red cells.
The low rate of freezing and the possibility to store at relatively high temperatures (below -65°C), makes the HGM the more practical cryopreservation method: cells can be frozen and stored in mechanical freezers and transported on “dry ice”. The obligatory use of liquid nitrogen precludes the LGM from being used in an operational, military setting, leaving the HGM as the only method to extend shelf lives of blood products for military purposes.
Cryoprotection by glycerol and its pretransfusion removal
Glycerol is most effective in protecting those cells into which it permeates fairly rapidly. Human erythrocytes have a high permeability for glycerol, unlike erythrocytes of pigs, dogs and cats for instance.37 Uptake of glycerol occurs both by active (or facilitated) and passive diffusion.38-40 The former is a metabolic process and as such temperature dependent. Passive diffusion is essentially independent of temperature.38 Glycerol and other intracellular cryoprotectants
form very firm hydrogen bonds with intracellular water, preventing this captured water turning into ice. With the available water for ice crystallization minimized, glycerol suppresses the rise in NaCl concentration, thereby avoiding extreme hypertonicity.41-43 Glycerol is thus effective in preventing cellular lysis due to osmotic damage during freezing and thawing.38
Regardless of the glycerol concentration used, postthaw washing of cryopreserved RBC is necessary to lower the concentration of glycerol to less than 1% (w/v) before transfusion.44 On contact with (isotonic) plasma, the glycerolized cells would otherwise hemolyze, because water enters the red cell faster than glycerol is able to move out.45This would cause swelling and eventually hemolysis if no precautions were taken. Different methods have been developed and used to achieve acceptable concentrations of glycerol in thawed red cell preparations, like dilution, dialysis and serial or continuous washing.
In 1954 the Cohn fractionator, originally designed to extract protein fractions from blood plasma, was modified to add and remove glycerol.46 The device used a continuous centrifugation process with a gradient hypertonic electrolyte wash of glycerol, sodium lactate and saline. Although successful, it proved to be a complicated, time consuming and impractical technique for widespread clinical use.47
In 1963 Huggins introduced a simpler method (without centrifugation) to remove glycerol, the so-called “reversible agglomeration”, to distinguish the
phenomenon from agglutination and aggregation.48 The procedure removes
the glycerol by adding large volumes of non-electrolyte solutions containing glucose and fructose, while stirring. As a result, the red cell environment is low in ionic strength and the cells start to clump spontaneously and settle as soon as stirring is stopped. The supernatant is then decanted and disaggregation of the retained red cell mass is then achieved by the addition of electrolyte solutions, like isotonic saline.41 Prior to transfusion, the red cells need to be concentrated to obtain a final hematocrit of 90%.49
Despite extensive and favorable (clinical) experience with this technique, gained at the US Naval hospital in Chelsea (MA)50-52 and, later, during the Vietnam War,53,54 the main source in the Vietnam War remained red cells prepared without freezing. However, between 1966 and 1969, the US Navy in Da Nang used more than 2,000 units of frozen red cells (O pos and O neg) with satisfactory results.55 The major logistic problems with the Huggins-method proved to be the large volume of washing solution and deglycerolization time.56
Chapter 1 Introduction Moreover, after storage at -80°C for 18 months or longer, the RBC recovery
values were only around 60%. Altogether, these drawbacks outweighed the shorter shelf life of the abundantly available fresh blood and the frozen blood banks were withdrawn from Vietnam in 1972. The unused frozen units were transported back to the US in styrofoam containers filled with dry ice and used for further research purposes.57
In 1967, the Arthur D. Little Company introduced the (reusable) stainless steel bowl,58 later followed by the (disposable) polycarbonate version in
several generations of Haemonetics blood processors.59 Both bowls were
used for continuous-flow washing and have been extensively tested by Valeri and associates.60-65 The M115 cell processor (Haemonetics, 1976), with a polycarbonate bowl, had an integrally attached shaker, to ensure adequate mixing of the various washing solutions. It was actually deployed and used during the first Gulf War (1990-1991). Deglycerolization took only about 35 minutes and 2L of washing fluids,66 in contrast to the Huggins method, taking 50 minutes and 6.8 L.55 It was still an open system, though, so the thawed red cells had to be administered within 24 hr after thawing and washing, in conjunction with storage at 2-6°C.
The practical use of frozen red cells during conflicts in the past two decades In 1990, during the build-up to the first Gulf War, some 7,000 frozen units were shipped to the area of conflict, of which only 265 were deglycerolized, without any transfusion. In sharp contrast, while preparing for this war during Desert Shield, some 82,000 liquid red cell units were shipped to the area of conflict, of which around 250 units were used for 250 US casualties and another 750 units for Iraqi prisoners of war (POW) and civilians. Ultimately, around 67,000 liquid red cell units, 80 percent of what was sent, were not used and had to be discarded.12 This outcome closely resembles the fate of the 3262 units of liquid red cells, provided to support the British armed forces in the Falklands War in 1982. Only 605 were used, giving a usage rate of 18.5 %.67
In 1991, the Netherlands military took part in the first Gulf War, mainly by deploying a navy task force, which was supplied with regular shipments of fresh red cells (2-6°C). The same array of products was used during the 1992-1993 United Nations-mission in Cambodia (UNTAC).
The experiences obtained from these two missions showed that worldwide deployments create serious logistic challenges, including those regarding blood
supply. Considerable distances need to be bridged between the Netherlands and the area of operations. Other than the fact that this takes precious time, the quantities and points in time where blood products are needed, cannot be predicted in wartime conditions. The relatively short shelf lives of particularly platelets and red cells would require frequent shipments. However, the points in time where and how much of the necessary blood products are needed would still remain unpredictable. So, even if at all times all necessary blood products would be made available, using the standard storage techniques, the operational needs would only be covered against considerable costs. At the same time, there would still be the danger of having to discard substantial numbers of units, due to outdating.12,67 This adds an ethical aspect to the discussion, because all blood products have been provided by volunteer, non-remunerated donors, aiming at helping those in need of transfusions. The only storage method, currently available, that guarantees the highest degree of self-sufficiency possible for the deployed military units, with the lowest outdating rates, is the application of cryopreservation. The first steps of the Netherlands military towards a blood supply system based on frozen blood products were taken during the UN- and NATO-missions in Bosnia (1992-1995 and 1995-2004 respectively), during which frozen red cells (using the HGM according to Valeri), FFP and eventually, by the end of 2001, frozen platelets in dimethyl sulfoxide (DMSO), all stored at - 80°C, became available to treat hemorrhaging war casualties. Deglycerolization of the thawed red cells was still performed with the (Haemonetics) M115, therefore allowing only for 24 hr of postthaw storage. After the introduction of the fully closed, automated cell processor ACP 215 in 1998, it became possible to extend the postthaw shelf life of thawed red cells to 14 days, also because of the application of AS-3 (Nutricel®) as the final storage solution.68 The Netherlands, taking part in missions in Iraq (2003-2005) and Afghanistan (2002-2014), gained extensive experience with shipping, storing, preparing and administering (previously) frozen blood products in war time conditions, achieving a safe and effective blood supply under wartime conditions, with a minimum wastage rate due to outdating and a minimal burden to the logistic system.69,70
In the next chapters the developments in the Military Blood Bank and research efforts to improve the practicality of the use of frozen red cells in battlefield (and civil) conditions will be discussed.
Chapter 1
20
Introduction
21
References
1. Kuhns WJ. Blood Transfusion in the Civil War. Transfusion 1965;5:92-4.
2. Schmidt PJ. Transfusion in America in the eighteenth and nineteenth centuries. N.Engl.J.Med. 1968;279(24):1319-20.
3. Schneider WH. Blood transfusion in peace and war, 1900-1918. Soc.Hist Med. 1997;10(1):105-26.
4. Mollison PL. The introduction of citrate as an anticoagulant for transfusion and of glucose as a red cell preservative. Br.J.Haematol. 2000 Jan;108(1):13-8.
5. Stansbury LG, Hess JR. Blood transfusion in World War I: the roles of Lawrence Bruce Robertson and Oswald Hope Robertson in the “most important medical advance of the war”. Transfus.Med.Rev. 2009;23(3):232-6.
6. Robertson LB. The transfusion of whole blood: a suggestion for its more frequent employment in war surgery. Br.Med.J. 1916;2(2897):38-40.
7. Pelis K. Taking credit: the Canadian Army Medical Corps and the British conversion to blood transfusion in WWI. J.Hist Med.Allied Sci. 2001;56(3):238-77.
8. Pelis K. Edward Archibald’s notes on blood transfusion in war surgery--a commentary. Wilderness.Environ.Med. 2002;13(3):211-4.
9. Crile G. I. The Technique of Direct Transfusion of Blood. Ann.Surg. 1907;46(3):329-32.
10. Giangrande PL. The history of blood transfusion. Br.J.Haematol. 2000;110(4):758-67.
11. Wise MW, O’Leary JP. The origins of blood transfusion: the later phase. Am.Surg. 2001;67(10):1011-3.
12. Hess JR, Thomas MJG. Blood use in war and disaster: lessons from the past century. Transfusion 2003;43(11):1622-33.
13. Rous P, Turner JR. The preservation of living red blood cells in vitro: I. Methods of preservation. J.Exp.Med. 1916;23(2):219-37.
14. Hess JR, Schmidt PJ. The first blood banker: Oswald Hope Robertson. Transfusion 2000;40(1):110-3.
15. Hanigan WC, King SC. Cold blood and clinical research during World War I. Mil. Med. 1996;161(7):392-400.
16. Schneider WH. Blood transfusion between the wars. J.Hist Med.Allied Sci. 2003;58(2):187-224.
17. Coni N. Medicine and the Spanish Civil War. J.R.Soc.Med. 2002;95(3):147-50. 18. Franco A, Cortes J, Alvarez J, Diz JC. The development of blood transfusion:
the contributions of Norman Bethune in the Spanish Civil War (1936-1939). Can.J.Anaesth. 1996;43(10):1076-8.
19. Pinkerton PH. Norman Bethune and transfusion in the Spanish Civil War. Vox Sang. 2002;83 Suppl 1:117-20.
20. Lethbridge D. “The blood fights on in other veins”: Norman Bethune and the transfusion of cadaver blood in the Spanish Civil War. Can.Bull.Med.Hist 2012;29(1):69-81.
21. Kendrick DB. Blood Program in World War II. 1989. 1964. Med Dep US Army. 22. Jeffrey HC. Blood Transfusion in War. J.R.Army Med Corps 1974;120:24-30. 23. Schmidt PJ, Huestis DW. Blood from cadavers: the final recycling. Transfusion
2007;47(4):555-6.
24. Grifols J. ISBT Science Series. 2007. p. 134-8.
25. Telischi M. Evolution of Cook County Hospital Blood Bank. Transfusion 1974;14(6):623-8.
26. Kevorkian J, Marra JJ. Transfusion of Human Corpse Blood Without Additives. Transfusion 1964;4:112-7.
27. Alexi-Meskishvili V, Konstantinov IE. Sergei S. Yudin: an untold story. Surgery 2006;139(1):115-22.
28. Huestis DW. Russia’s National Research Center for Hematology: its role in the development of blood banking. Transfusion 2002;42(4):490-4.
29. Hardaway RM. 200 years of military surgery. Injury 1999;30(6):387-97.
30. Standards for Blood Banks and Transfusion Services. 30th edition. 2016. AABB. 31. Guide to the Preparation, Use and Quality Assurance of Blood Components. 18th
edition. 2015. Strasbourg, Council of Europe.
32. Valeri C.R.and Gina Ragno Giorgio. The US Navy’s experience with resuscitation of wounded servicemen in Vietnam using frozen washed red blood cells from 1966 to 1974: developments from this experience. In Forty-five years of Research at the NBRL, Boston, Massachusetts. first ed. 2013. p. 197.
33. Smith AU. Prevention of haemolysis during freezing and thawing of red blood-cells. Lancet 1950;2(6644):910-1.
34. Pert JH, Schork PK, Moore R. Low-temperature preservation of human erythrocytes: biochemical and clinical aspects. Bibl.Haematol. 1964;19:47-53. 35. Krijnen HW, De Wit JJ, Kuivenhoven AC, Loos JA, Prins HK. Glycerol treated
human red cells frozen with liquid nitrogen. Vox Sang. 1964;9:559-72.
36. Rowe AW, Eyster E, Kellner A. Liquid nitrogen preservation of red blood cells for transfusion; a low glycerol-rapid freeze procedure. Cryobiology 1968;5(2):119-28. 37. Yaeger Y, Nathan I, Dvilansky A, Meyerstein N. Permeability of fresh and
stored human erthrocytes to glycerol and its acylated derivatives. Experientia 1979;35(12):1673-4.
38. Huggins CE. Frozen blood: principles of practical preservation. Monogr Surg.Sci. 1966;3(3):133-73.
39. Carlsen A, Wieth JO. Glycerol transport in human red cells. Acta Physiol Scand. 1976;97(4):501-13.
40. Roudier N, Verbavatz JM, Maurel C, Ripoche P, Tacnet F. Evidence for the presence of aquaporin-3 in human red blood cells. J.Biol.Chem. 1998;273(14):8407-12. 41. Huggins CE. Red Cell Freezing; A Technical Workshop. In 1973 Nov 11; AABB;
1973. p. 31-53.
42. Rall WF, Mazur P, Souzu H. Physical-chemical basis of the protection of slowly frozen human erythrocytes by glycerol. Biophys.J. 1978;23(1):101-20.
Chapter 1 Introduction
43. Pegg DE, Diaper MP. The effect of initial tonicity on freeze/thaw injury to human red cells suspended in solutions of sodium chloride. Cryobiology 1991;28(1):18-35.
44. Valeri CR. Recent advances in techniques for freezing red cells. CRC Crit Rev.Clin. Lab Sci. 1970;1(3):381-425.
45. Armitage WJ. Metabolism and Physiology of Cells at Low Temperatures. Cryopreservation and Low Temperature Biology in Blood Transfusion[24], 1-10. 1989. Kluwer Academic Publishers.
46. Tullis JL, Surgenor DM, Tinch R, D’Hont M, Gilchrist FL, Driscoll S, Batchelor WH. New principle of closed system centrifugation. 1956. Ther.Apher. 2000;4(2):73-80.
47. Valeri CR. Preservation of human red blood cells. Bull.N.Y.Acad.Med. 1968;44(1):3-17.
48. Huggins CE. Reversible agglomeraton used to remove dimethylsulfoxide from large volumes of frozen blood. Science 1963;139(3554):504-5.
49. Valeri CR. A comparison of washing methods. In Blood Banking and the Use of Frozen Blood Products. CRC Press; 1976. p. 178.
50. Valeri CR, Bond JC, McCallum LE. Relationships between metabolic state and (1) in vivo survival and (2) density distribution of previously frozen human erythrocytes. Transfusion 1966;6(6):543-53.
51. Almond DV, Valeri CR. The in vivo effects of deglycerolized agglomerated erythrocytes transfused in multiple units to stable anemic patients. Transfusion 1967;7(2):95-104.
52. Valeri CR, Runck AH, McCallum LE. Observations on autologous, previously frozen, deglycerolized, agglomerated, resuspended red cells. I. Effect of storage temperatures. II. Effect of adenine supplementation of glycerolized red cells prior to freezing. Transfusion 1967;7(2):105-16.
53. Valeri CR, Brodine CE, Moss GE. Use of frozen blood in Vietnam. Bibl.Haematol. 1968;29:735-8.
54. Moss GS, Valeri CR, Brodine CE. Clinical experience with the use of frozen blood in combat casualties. N.Engl.J.Med. 1968;278(14):747-52.
55. Valeri C.R.and Gina Ragno Giorgio. The US Navy’s experience with resuscitation of wounded servicemen in Vietnam using frozen washed red blood cells from 1966 to 1974: developments from this experience. In Forty-five years of Research at the NBRL, Boston, Massachusetts. first ed. 2013. p. 208-9.
56. Valeri C.R.and Gina Ragno Giorgio. The US Navy’s experience with resuscitation of wounded servicemen in Vietnam using frozen washed red blood cells from 1966 to 1974: developments from this experience. In Forty-five years of Research at the NBRL, Boston, Massachusetts. first ed. 2013. p. 204.
57. Valeri CR. Factors which affect the therapeutic effectiveness of red cells freeze-preserved with glycerol. In Blood Banking and the Use of Frozen Blood Products. CRC Press; 1976. p. 54.
58. Tullis JL, Tinch RJ, Gibson JG, Baudanza P. A simplified centrifuge for the separation and processing of blood cells. Transfusion 1967;7(3):232-42.
59. Tullis JL, Gibson JG, Tinch RJ, Hinman J, Baudanza P, DiForte S, Smith T, Breed AT. Disposable plastic centrifuge bowls for separation of red blood cells and plasma in the processing of frozen blood. Transfusion 1971;11(6):358-67.
60. Runck AH, Valeri CR, Sampson WT. Comparison of the effects of ionic and non-ionic solutions on the volume and intracellular potassium of frozen and non-frozen human red cells. Transfusion 1968;8(1):9-18.
61. Valeri CR, Runck AH. Long term frozen storage of human red blood cells: studies in vivo and in vitro of autologous red blood cells preserved up to six years with high concentrations of glycerol. Transfusion 1969;9(1):5-14.
62. Runck AH, Valeri CR. Recovery of glycerolized red blood cells frozen in liquid nitrogen. Transfusion 1969;9(6):297-305.
63. Crowley JP, Valeri CR. The purification of red cells for transfusion by freeze preservation and washing. I. The mechanism of leukocyte removal from washed, freeze-preserved red cells. Transfusion 1974;14(3):188-95.
64. Crowley JP, Valeri CR. The purification of red cells of transfusion by freeze preservation and washing. II. The residual leukocytes, platelets, and plasma in washed,freeze-preserved red cells. Transfusion 1974;14(3):196-202.
65. Crowley JP, Valeri CR. The purification of red cells for transfusion by freeze-preservation and washing. III. Leukocyte removal and red cell recovery after red cell freeze-preservation by the high or low glycerol concentration method. Transfusion 1974;14(6):590-4.
66. Valeri CR, Valeri DA, Anastasi J, Vecchione JJ, Dennis RC, Emerson CP. Freezing in the primary polyvinylchloride plastic collection bag: a new system for preparing and freezing nonrejuvenated and rejuvenated red blood cells. Transfusion 1981;21(2):138-49.
67. Marsh AR. A short but distant war - the Falklands campaign. J.R.Soc Med 1983;76(11):972-82.
68. Valeri CR, Ragno G, Pivacek LE, Srey R, Hess JR, Lippert LE, Mettille F, Fahie R, O’Neill EM, Szymanski IO. A multicenter study of in vitro and in vivo values in human RBCs frozen with 40-percent (wt/vol) glycerol and stored after deglycerolization for 15 days at 4 degrees C in AS-3: assessment of RBC processing in the ACP 215. Transfusion 2001;41(7):933-9.
69. Lelkens CCM, Koning JG, de Kort B, Floot IBG, Noorman F. Experiences with frozen blood products in the Netherlands military. Transfus.Apher.Sci. 2006;34(3):289-98.
70. Noorman F, van Dongen TTCF, Plat MC, Badloe JF, Hess JR, Hoencamp R. Transfusion: -80 degrees C Frozen Blood Products Are Safe and Effective in Military Casualty Care. PLoS.One. 2016;11(12):e0168401
Charles C.M. Lelkens1, Femke Noorman1, Jack G. Koning1, Rosa Truijens-de Lange2, Perry S. Stekkinger2, Joa C. Bakker2, Johan W.M. Lagerberg2, Anneke Brand3 and Arthur J. Verhoeven2
1Military Blood Bank, Leiden, 2Department of Transfusion Technology, Sanquin Research at CLB, Amsterdam, 3Blood Bank Sanquin South-West, Leiden
Transfusion. 2003;43(2):157-64
Chapter
Stability after thawing of RBCs frozen with the
high- and low-glycerol method
Chapter 2 Stability after thawing of RBCs frozen with the high- and low-glycerol method
Abstract
Background: RBCs can be frozen with either the high-glycerol method (HGM) or the low-glycerol method (LGM). To date, the use of frozen RBCs is hampered by a 24 hour outdating period after thawing. A closed washing system (ACP 215) may solve this problem.
Study design and methods: We compared the effects of high- (40%) and low-glycerol (19%) concentration, with and without freezing (at -80°C for HGM, -196°C for LGM) on the in vitro quality of RBCs after deglycerolization with the closed washing system and during storage at 4°C in SAGM after thawing.
Results: Glycerol treatment by itself induced hemolysis during processing, which was more pronounced in HGM cells. The freeze-thaw-wash process decreased the stability of RBCs, particularly in LGM cells during storage after thawing. In contrast to LGM cells, in HGM cells no additional effect of freeze or thaw on stability of washed cells was seen during the first week of storage after thawing. Changes in osmotic resistance and cellular metabolism could not explain the observed differences in RBC stability.
Conclusion: The closed washing system is able to process both high- and low-glycerol-treated RBCs. Stability after washing during cold storage in SAGM, as measured by hemolysis, is better for HGM cells as compared to LGM cells.
Introduction
Freezing RBCs with glycerol as a cryoprotectant dates back to 19501 after an accidental discovery the previous year.2 In the 1960s, it was found that accelerating the rate of freezing could significantly reduce the required concentration of glycerol to 19-20%.3-5After several modifications6-9 the two methods, referred to as the high- (HGM) and low-glycerol methods (LGM) respectively, are still in use for freezing units of RBCs. HGM allows storage at -80°C, whereas LGM requires liquid nitrogen (-196°C). Some laboratories prefer LGM over HGM because of a shorter processing time and a more or less indefinite shelf life at temperatures below -130°C.10,11Recently, however, Valeri et al.12 have shown that cells frozen with HGM and stored at - 80°C can be stored for up to at least 37 years with an acceptable recovery after thawing and washing. Despite their undeniably higher costs,13-16 frozen RBCs have several advantages that are, paradoxically, mainly related to the necessary washing procedure, eliminating cell debris, WBCs, cytokines, and free Hb.17-19 Stockpiling a frozen inventory of RBCs can be done in the case of rare blood groups and for military deployments, which characterized by logistical problems and unpredictable needs of blood components.20 However, besides cost, the actual use of frozen RBCs is hampered mainly by processing time and a 24 hour outdating period due to potential bacterial contamination if thawing and washing is performed in a non-closed system.21,22 Recently, a fully automated and functionally closed washing system for RBCs has been introduced (ACP 215, Haemonetics, Braintree, MA), which may contribute to a solution of these problems.
Although a closed washing system has been developed for processing RBCs according to HGM,23-25 we investigated its application for LGM as well. In a paired in vitro study, we compared the quality of the final RBCs using HGM and LGM, and we determined whether differences in quality were related to differences in glycerol concentration or to the freeze-thaw process.
Materials and methods
Whole blood donations and processingWBC reduction of blood components to less than 1x106 WBCs per unit is
Chapter 2 Stability after thawing of RBCs frozen with the high- and low-glycerol method
28 29
95%,17,26,27 which is still above the limit.24,25 Therefore, we used filtered whole blood as starting material in this study.
The filtered units were subsequently pooled and split. After plasma removal, the units were either stored at 4⁰C in SAGM or treated with 40% or 19% glycerol (Fig. 1). The glycerolized RBCs were either immediately washed or frozen to -80°C or -196°C, respectively, and washed after thawing. We used a sterile connection device (model SC-201 AH, Terumo Europe, Leuven, Belgium) to perform all necessary tube welding. The washed cells were stored for 36 days at 4°C in SAGM.
In detail, 20 volunteers, who met AABB requirements for blood donors and provided informed consent, donated whole blood (500 mL, 66 ± 1 g of Hb) into a 600 mL bag (NPBI-Fresenius, Emmer-Compascuum, the Netherlands) containing 70 mL of CPD. Within 18 hours after collection and storage at room temperature, the units were WBC depleted (Imuflex, Whole Blood Filter, Terumo, Tokyo, Japan), which resulted in units containing 61 ± 2 g of Hb (a loss of 5.4 ± 1.6 g of Hb). After WBC depletion, we pooled 5 units in a 2 L bag
(PL 1813/1, Baxter, Deerfield, IL) and, after sampling, split the pool into 5 equal units (58 ± 4 g of Hb), using 800 mL bags (PL 146, Baxter, Deerfield, IL). The units were centrifuged for 4 minutes at 1615 x g, supernatant plasma was
removed, and aliquots of plasma were stored at -80°C for plasma stability tests (see below).
The units of RBCs were treated in five different ways (Fig. 1). Total processing time took about 22 hours at room temperature from the time of donation until storage at the indicated temperatures. One unit (WBC depleted, Hct ∼ 0.80) was diluted with SAGM (Haemonetics) to an Hct of approximately 0.43, split into 8 parts of 30 mL in PVC bags (100 mL, Compoflex, NPBI-Fresenius, Emmer Compascuum, the Netherlands), and subsequently stored at 4°C. The four other units of the pool were treated with either 40% or 19% glycerol. The cells treated with glycerol were either directly deglycerolized as described below, using an automated closed washing system (ACP 215, Haemonetics) or frozen to -80°C or -196°C, respectively. After being frozen for 4 to 6 weeks, we thawed and deglycerolized these units. Approximately 4 g of Hb was lost due to glycerolization, sampling, bag transfer, and removal of supernatant glycerol. On average, 54 ± 4 g of Hb was recovered in the waste bag and component bag after thawing and deglycerolization with the closed washing system. The cells were resuspended in SAGM during the last deglycerolization step (Hct 0.50 ± 0.02, n = 16), split into 8 parts of 30 mL, and stored in PVC bags at 4°C. At different times during storage (as indicated in the figures), we took the PVC bags out of the refrigerator for sampling.
Glycerolization
Briefly, 38% wt/vol glycerol (containing 2.9% wt/vol sorbitol and 0.63% wt/ vol NaCl, NPBI) was added manually in about 2 minutes to an equal volume of RBCs (Hct ∼ 0.80) to obtain a final concentration of 19% glycerol for LGM. For HGM, 57% wt/vol glycerol (containing 1.6% sodium lactate, 0.03%
KCl, 0.0517% Na2HPO4, 0.1242% NaH2PO4, pH 6.8; Baxter) was added
with the closed washing system in about 10 minutes to RBCs (Hct ∼ 0.80), proportionally to unit weight (a modifiable parameter of the ACP 215) to obtain a final concentration of 40% glycerol. Osmolality change during this process was kept at a constant rate of 500 mOsm per kg per minute.
Fig. 1. Schematic overview of study design
Leuko Filter Filtered blood Plasma RBCs 19% glycerol (LGM) Deglycerolization ACP215 (50 mL 6%NaCl 1870 mL 0.9%NaCl) RBC+SAGM 4°C control
Pool with 5 units filtered blood;
split centrifuged separation of plasma and RBCs Whole Blood donation RBCs RBCs RBCs RBCs RBCs 40% glycerol (HGM) 19% glycerol (LGM) Deglycerolization ACP215 (50 mL 12%NaCl 1870 ml 0.9%NaCl) Deglycerolization ACP215 (50 mL 6%NaCl 1870 mL 0.9%NaCl) Deglycerolization ACP215 (50 mL 12%NaCl 1870 mL 0.9%NaCl) -196°C 6 weeks - 80°C 6 weeks 40% glycerol (HGM) RBC+SAGM 4°C LGM non-frozen RBC+SAGM 4°C HGM non-frozen RBC+SAGM 4°C LGM frozen RBC+SAGM 4°C HGM frozen Removal supernatant glycerol Removal supernatant glycerol Removal supernatant glycerol
Chapter 2 Stability after thawing of RBCs frozen with the high- and low-glycerol method Freezing and thawing
The LGM-treated cells (Hct ∼ 0.40) were transferred to an aluminum container (600 mL) (Boxal, Veenendaal, the Netherlands) and frozen within 10 minutes in liquid nitrogen.4,28 HGM-treated cells were centrifuged to remove excess glycerol (10 min at 1248 x g, final Hct ∼ 0.70). The PVC bag (Baxter) containing the RBCs was sealed in a plastic overwrap, placed in a cardboard box (Cekumed, Ooltgensplaat, the Netherlands), and put on the bottom of a -80°C freezer, inducing a slow drop in temperature (1-3°C/min) to -80°C.9 Thawing in a 37°C water bath took about 10 minutes and 20 minutes for LGM and HGM frozen RBCs, respectively. All units were thawed and warmed to a temperature of 30°C. Before deglycerolization, excess glycerol in the LGM units was removed by centrifugation at 2250 x g for 5 minutes.
Deglycerolization
The closed washing system (ACP 215) was developed and FDA approved for the glycerolization and deglycerolization of HGM cells.24,25 For LGM, we developed a new deglycerolization procedure that would fit the programming of the closed washing system (ACP 215), washing fluids, and disposables (see below). For this new procedure, we compared the components obtained from the standard
LGM procedure28 with the components obtained after introduction of the
changes described below, using standard frozen LGM units.28 In the first washing step with hypertonic solution, 17.5% (wt/vol) sorbitol was replaced by a smaller volume of 6% (wt/vol) saline to make this step analogous to the addition of 12% (wt/vol) saline in HGM (see below) and to avoid volume overload in the bag. Secondly, we compared the current discontinuous washing procedure with a continuous washing procedure (carried out on an M 115 cell washer; Haemonetics Corp., Braintree, MA). These changes not only promoted recovery after the deglycerolization procedure (6% saline vs. 17.5% sorbitol) but also slightly shortened the processing time (continuous vs. discontinuous washing) and, hence, were considered to be appropriate changes to LGM (results not shown).
In the current study the standard programming of the closed washing system was used for deglycerolization of both LGM and HGM cells. For the deglycerolization of cells treated with 40% glycerol, 50 mL of 12% (wt/vol) saline was added to the glycerolized (and thawed) cells. For the deglycerolization of cells treated with 19% glycerol, 50 mL of 6% (wt/vol) saline was added. The
rest of the closed washing system deglycerolization procedure was completely the same for both LGM- and HGM-treated cells. After an incubation period of 2.5 minutes, 340 mL of 0.9% (wt/vol) saline and 0.2% (wt/vol) glucose was added. The cells were incubated for 1 minute. The diluted cells were subsequently pumped to the bowl, and supernatant saline was removed during centrifugation and pumped to the waste bag. The RBCs were returned to the original bag with an additional volume of a fresh 50 mL of 0.9% (wt/vol) saline and 0.2% (wt/ vol) glucose to rinse the bowl. The cells were then diluted a second time with 400 mL of 0.9% (wt/vol) saline and 0.2% (wt/vol) glucose. After 1 minute of incubation, the diluted cells were pumped to the bowl and washed with 1080 mL of 0.9% (wt/vol) saline and 0.2% (wt/vol) glucose. In the final step, the cells were washed with, and resuspended in, 240 mL of SAGM and pumped to the storage bag. On average, the total closed washing system procedure of 1 unit took 65 ± 7 minutes (mean ± SD, n = 16 procedures).
Hb content and hemolysis during processing
The washing fluids collected in the waste bags and all storage and component bags were sampled to determine the total amount of Hb and the amount of Hb in supernatants of the samples after centrifugation for 10 minutes at 1400 x g. Hb was measured using the cyanmethemoglobin method.29 Hemolysis was expressed as a percentage of total Hb by using the following formula:
Hemolysis = 100% x {(supernatant Hb) x (supernatant volume) / {(total Hb) x (total volume)}
Plasma stability and osmotic resistance
We used two different concentrations of saline to estimate osmotic resistance. Samples (50 μL) at days 1 and 36 of units were diluted in 2 mL of 0.1% (wt/ vol) Triton X-100 and 0.001% (wt/vol) saponin, 0.5% (wt/vol) saline, 0.6% (wt/vol) saline, 0.9% (wt/vol) saline or in autologous pool plasma. These samples were incubated for 30 minutes at 22°C and subsequently centrifuged at 1400 x g for 10 minutes. To meet the required increased sensitivity, Hb in
the supernatant was measured directly on a spectrophotometer (Spectronic 301, Milton Roy, Ivyland, PA) using wavelengths 542 an 415 nm for samples in saline and 600, 577, 542, 510, 415, and 370 nm for samples in plasma.30 Hemolysis was calculated using the following formula:
Chapter 2 Stability after thawing of RBCs frozen with the high- and low-glycerol method
32 33
RBC density distribution
Samples of days 1 and 36 stored units were centrifuged at 1400 x g for 10 minutes,
supernatant was removed, and 6 mL of RBCs were mixed with 25 mL of a Percoll solution (323 mOsmol/L, pH 8.24), slightly modified from Lutz et al.31 1 L of buffered Percoll solution contained 0.0372 g EDTA, 1.00 g D-glucose, 1.332 g NaCl, 0.356 g Na2HPO4, and 948 g Percoll (Pharmacia, Uppsala, Sweden).
The Percoll and RBCs were mixed and centrifuged at 43000 x g for 20
minutes at 16 to 23°C. The self-formed Percoll gradient with layers of RBCs was aspirated at a rate of 1 mL per minute from the bottom of the tube. Fractions of 3.5 mL were collected, and Hb content was measured in each fraction using the
cyanmethemoglobin method.29
RBC metabolism
Samples (0.5 mL) of the various RBC suspensions were diluted 1 in 1 in SAGM and deproteinized by adding 0.3 mL of 14% (wt/vol) perchloric acid.32,33 After 10 minutes on ice, samples were centrifuged and the protein-free supernatant was neutralized with ice-cold 2 N KOH and 0.2 mol MOPS per L. Neutralized extracts were subsequently stored at -80°C.
Adenine nucleotides were analyzed by high-performance liquid
chromatography (HPLC), as described by De Korte et al.32,33 Lactate was
determined enzymatically in these neutralized perchloric acid extracts by lactate oxidase and hydrogen peroxide activity (Sigma Chemical, St. Louis, MO). DPG was measured enzymatically using the combined action of 2,3-DPG phosphatase, phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase according to the manufacturer’s instructions (Boehringer, Mannheim, Germany). Neutralized perchloric acid extracts were also used to determine the residual concentration of glycerol. Glycerol was measured enzymatically using the combined action of glycerokinase, pyruvate kinase, and lactate dehydrogenase (Diffchamb-Biocontrol, Nieuwerkerk aan den IJssel, the Netherlands).
Statistics
Data were analyzed using software (Microsoft Excel, Bellevue, WA) and the paired Student’s t-test. In general, results are depicted as means ± SD of the
number of observations given in parentheses. A p- value of less than 0.05 was
considered significant.
Results
DeglycerolizationAs shown in Fig. 2, glycerolization of RBC units followed by deglycerolization without freezing already caused significant hemolysis. LGM cells showed considerably less hemolysis (2.4 ± 0.4%, n = 4) during deglycerolization than HGM cells (11 ± 3%, n = 4). The freeze-thaw process did not further affect hemolysis during deglycerolization of HGM RBCs (10 ± 2%, n = 4), whereas it almost doubled hemolysis during deglycerolization of LGM RBCs (4.4 ± 1.0%, n = 4). Clearly, total hemolysis during washing was significantly lower when LGM was used as compared to HGM.
Fig. 2. Hemolysis during deglycerolization. Hb content of the waste fluid supernatant was measured and expressed as a percentage of the total Hb content of the thawed unit before deglycerolization. Q, LGM method; O, HGM method.
0 2 4 6 8 10 12 14
Without freezing Frozen/thawed
He moly sis duri ng de gly ce roli za tion (% )
Chapter 2 Stability after thawing of RBCs frozen with the high- and low-glycerol method The final Hb content of the deglycerolized LGM and HGM units showed no
difference because this parameter was mainly determined by the (limited) bowl volume, which we found to be 170 mL of RBCs. Volumes of RBCs exceeding this limit lead inevitably to cell spillage. Cell spillage was highest with the LGM cells (7 ± 5%) compared to HGM cells (4 ± 3%). Consequently, all our procedures yielded deglycerolized components with a cellular Hb content exceeding 36 g per unit (48 ± 2 g of Hb/ unit, n = 16) and free Hb below 0.2 g per unit (0.13 ± 0.05 g/unit, n = 16) in conformance with European guidelines.22The remaining glycerol concentrations were 0.02 ± 0.01% wt/vol in our LGM samples and 0.04 ± 0.01% wt/vol in the HGM samples, all well below the critical residual glycerol concentration of 1 to 2%.6,14,19,20 There was no difference between frozen and non-frozen units, and glycerol concentrations remained the same during storage (results not shown).
Storage after washing
In the final washing step, the cells were resuspended in SAGM. Immediately after processing, hemolysis did not differ between treatments (0.28 ± 0.10%, n = 16; Fig. 3). However, hemolysis was significantly higher in all LGM and HGM cells compared to the untreated control RBCs (0.11 ± 0.05%, n = 4; Fig. 3).
During storage from days 1 to 36, glycerol treatment without freezing increased hemolysis. This effect of glycerol on RBCs was similar with both glycerol concentrations (Fig. 3).
In frozen and thawed cells, however, a pronounced difference was observed between the two methods. From day 1 through day 8 of storage, hemolysis in LGM (-196°C) cells was significantly higher than in HGM (-80°C) cells (Fig. 3). This difference between the two freeze-thaw procedures disappeared after day 15 of storage.
Osmotic resistance and plasma stability
To evaluate osmotic RBC resistance after storage in SAGM, hemolysis was measured on days 1 and 36 of storage. On day 1, we hardly saw an effect of treatment, whereas on day 36, a reduction of osmotic resistance due to deglycerolization and freezing and thawing was observed. Both on day 1 and day 36, hemolysis in plasma was 60 percent lower than in 0.9% saline.
No differences were observed between HGM and LGM cells.
RBC density distribution
Hemolysis during deglycerolization was relatively high with HGM RBCs (Fig. 2) but relatively low during storage after thawing (Fig. 3). The higher stability after thawing of frozen and thawed HGM cells may have been due to selection of a population of relatively strong (and young) cells. Differences in RBC density have been attributed to RBC aging.31 We therefore studied cell density on days 1 and 36 of storage using a continuous Percoll gradient.31 No significant change in density distribution was noted among any of the suspensions tested. Compared to day 1 of storage, we did measure a significant increase of cells in the highest
Fig. 3. Hemolysis during storage in SAGM after wash. Component was stored for 35 days at 4°C in SAGM. On the indicated time points, the Hb content of the component supernatant was measured and expressed as a percentage of the total Hb content of the component. F, control cells; , LGM non-frozen cells; , LGM frozen cells; G, HGM non-frozen cells; O , HGM frozen cells.
Chapter 2 Stability after thawing of RBCs frozen with the high- and low-glycerol method
36 37
density fraction of cells after day 36 of storage (from 0.7 to 1.4%), but, again, there was no significant difference between treatments. This indicates that, if these high-density cells are aged RBCs, we could not detect selective removal of these older cells.
RBC metabolism
To explain the lower stability after thawing of LGM cells, several parameters of RBC metabolism were measured. The 2,3-DPG content of the control cells was already very low after day 1 of storage (3.9 ± 1.1 μmol/g of Hb, n = 4) and below the detection limit after day 2 of storage. The 2,3-DPG content appeared to be somewhat lower in the glycerol-treated cells, but these differences were not significant (results not shown).
Production of lactate did not differ between treatments during the first 8 days of storage, but from day 15 the control cells showed a significantly higher lactate production (on day 15 control cells 90 ± 12 μmol/g of Hb vs. glycerol treated cells 61 ± 9 μmol/g of Hb).
As shown in Fig. 4A the ATP content of the cells was not significantly affected by the treatments during the first two days of storage. Thereafter, ATP was significantly lower in the glycerol-treated cells. As was observed for lactate production, there was no difference among the various treatments. We also measured ADP and AMP to determine the total adenylate energy charge of the stored cells, because this parameter shows a better correlation with survival than just ATP.34 The ADP content and AMP content were significantly higher in the glycerol treated cells from day 1 on. There was no difference in the ADP content between the treatments, whereas the AMP content was significantly higher in the LGM and HGM frozen cells from day 8 on. From these data the total adenylate charge (ATP + 0.5 ADP)/(ATP + ADP + AMP) was calculated, which showed that the glycerol-treated cells had a lower net energy charge from day 1 on (Fig. 4B). On day 15, the frozen cells showed a significantly lower energy charge as compared to the non-frozen cells. None of these parameters showed a significant difference between LGM and HGM cells.
Fig. 4. Energy content during storage in SAGM after wash. Component was stored for 35 days at 4°C in SAGM. On the indicated time points, the ATP, ADP, and AMP content of the cells was measured, expressed as μmol per g of Hb, and from this the adenylate charge was calculated. (A) ATP content; (B) adenylate charge. F, control cells; , LGM non-frozen cells;, LGM frozen cells; G , HGM non-frozen cells; O , HGM frozen cells. 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 95 90 85 80 75 70 65 5.5 5.0 4.5 4.0 3.5 3.0 2.5 Adenylate charge (%) ATP content (μmol/grHb)
Storage time at 4°C (days)
B A
Storage time at 4°C (days)
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 95 90 85 80 75 70 65 5.5 5.0 4.5 4.0 3.5 3.0 2.5 Adenylate charge (%) ATP content (μmol/grHb)
Storage time at 4°C (days)
B A
Chapter 2 Stability after thawing of RBCs frozen with the high- and low-glycerol method
Discussion
In this study, we compared two established methods for the cryopreservation of RBC using the newly developed washer (ACP 215) for deglycerolization. With the development of this fully automated and functionally closed washing system, it is now possible to deglycerolize RBCs while maintaining sterility, allowing subsequent storage beyond 24 hours.23-25 We studied the application of this washer for RBCs cryopreserved with LGM, and we compared the stability of these cells (during washing and subsequent storage) with RBCs prepared according to HGM.
Our study showed that by applying LGM, little hemolysis occurred during deglycerolization on the closed washing system and that freezing almost doubled this hemolysis. In contrast, the reverse was observed in HGM cells, in that more Hb was lost due to hemolysis during deglycerolization, whereas freezing hardly had an additional effect (Fig. 2). Hemolysis immediately after thaw and wash was similar for both LGM and HGM cells with or without freezing, showing that all free Hb was removed by the closed washer system deglycerolization procedure. Though relatively small numbers of units were used for the current study, the component loss due to hemolysis we observed during the LGM and HGM procedures is comparable to the losses (HGM 10-20%, LGM 5-10%) reported by others using the same24,25 or different deglycerolization procedures.5,9,15,17,24-28,35-38 All processed units met US and European guidelines for thawed deglycerolized RBCs.21,22 From these results it can be concluded that the closed washing system (ACP 215) can successfully be applied, not only to HGM, but also to LGM. However, to fully exploit the advantage of the washer to LGM, a liquid nitrogen resistant cryopreservation bag, suitable for sterile docking onto the washing device, is required to enable prolonged storage after washing beyond 24 hours.
Interestingly, the decreased stability of RBCs during washing caused by the higher glycerol concentration was not reflected in a decreased stability during storage after washing. Actually, during the first week of storage, hemolysis was highest for LGM frozen cells (Fig. 3). This lower stability after thawing of LGM cells was not due to the processing on the closed washing system because we observed similar differences in stability when processed by our current routine procedures.39 Two alternative explanations were considered to account for the lower stability after thawing of LGM cells. Firstly, the higher hemolysis during storage of LGM cells could be due to the preservation of a more labile (and
aged) population of RBCs. However, the density distributions of the different concentrates did not support this hypothesis. Alternatively, the lower hemolysis of HGM cells during storage after thawing could reflect a better protection against cell damage induced by the freeze and thaw process. This is indirectly supported by our finding that hemolysis in HGM cells during washing and during the first week of storage was not affected by freezing. Furthermore, hemolysis of HGM frozen cells was lower during storage as compared to LGM frozen cells. On the other hand, this improved protection by high glycerol leads to a higher degree of hemolysis during washing, most probably inflicted by a higher degree of osmotic changes as compared to LGM.
Other than hemolysis, no other parameter studied showed differences between the frozen HGM and the frozen LGM cells within the first week of storage. In contrast to previously published reports,14,40 we observed strongly reduced 2,3-DPG levels in all concentrates already at day 1. It was previously recognized that holding fresh whole blood for 24 hours at room temperature reduces 2,3-DPG levels from 13.1 to 4.4 μmol per g of Hb.41 In our experiments, the average holding time at room temperature between donation and time of storage was 22 hours. This may explain the above-mentioned 2,3-DPG levels.
In accord with data from Moroff and Meryman,42 we demonstrated that
all glycerolization and deglycerolization procedures with or without freezing resulted in a final product with a normal ATP content per cell and that this ATP level was maintained during the first 2 days of storage (Fig. 4A). However, we also showed that the adenylate content of the cells on the first day of storage was significantly lowered by the various treatments (Fig. 4B). Because the total adenylate content shows a better correlation with survival than just ATP,34 this may predict a lower “in vivo” survival of these SAGM-stored thawed and washed cells.
Similar to other investigators, we have found that, compared to SAGM, both previously frozen HGM and LGM RBCs demonstrated a reduced hemolysis in AS-3.23,37,39 “In vivo” recovery of the closed washing system-processed HGM cells, stored for 15 days in AS-3, appeared to be satisfactory;24,25 for LGM cells, this remains to be shown.
Chapter 2 Stability after thawing of RBCs frozen with the high- and low-glycerol method
40 41
Acknowledgments
The authors thank Dirk de Korte, PhD, and Eric Gouwerok (Sanquin Research at CLB, Amsterdam, the Netherlands) for their help with adenine nucleotide determinations.
References
1. Smith AU. Prevention of haemolysis during freezing and thawing of red blood-cells. Lancet 1950;2(6644):910-1.
2. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949;164(4172):666.
3. Pert JH, Schork PK, Moore R. Low-temperature preservation of human erythrocytes: biochemical and clinical aspects. Bibl.Haematol. 1964;19:47-53. 4. Krijnen HW, De Wit JJ, Kuivenhoven AC, Loos JA, Prins HK. Glycerol treated
human red cells frozen with liquid nitrogen. Vox Sang. 1964;9:559-72.
5. Rowe AW, Eyster E, Kellner A. Liquid nitrogen preservation of red blood cells for transfusion; a low glycerol-rapid freeze procedure. Cryobiology 1968;5(2):119-28. 6. Valeri CR, Brodine CE. Current methods for processing frozen red cells.
Cryobiology 1968;5(2):129-35.
7. Meryman HT, Hornblower M. A method for freezing and washing red blood cells using a high glycerol concentration. Transfusion 1972;12(3):145-56.
8. Valeri CR. Simplification of the methods for adding and removing glycerol during freeze-preservation of human red blood cells with the high or low glycerol methods: biochemical modification prior to freezing. Transfusion 1975;15(3):195-218. 9. Valeri CR, Valeri DA, Anastasi J, Vecchione JJ, Dennis RC, Emerson CP. Freezing
in the primary polyvinylchloride plastic collection bag: a new system for preparing and freezing nonrejuvenated and rejuvenated red blood cells. Transfusion 1981;21(2):138-49.
10. Mazur P. Limits to life at low temperatures and at reduced water contents and water activities. Orig.Life 1980;10(2):137-59.
11. Mazur P. Stopping biological time. The freezing of living cells. Ann.N.Y.Acad.Sci. 1988;541:514-31.
12. Valeri CR, Ragno G, Pivacek LE, Cassidy GP, Srey R, Hansson-Wicher M, Leavy ME. An experiment with glycerol-frozen red blood cells stored at -80 degrees C for up to 37 years. Vox Sang. 2000;79(3):168-74.
13. Valeri CR, Runck AH, Brodine CE. Recent advances in freeze-preservation of red blood cells. JAMA 1969;208(3):489-92.
14. Valeri CR. Recent advances in techniques for freezing red cells. CRC Crit Rev.Clin. Lab Sci. 1970;1(3):381-425.
15. Moss GS. Preservation of red cells by freezing. Surg.Annu. 1970;2(0):35-50. 16. Chaplin HJ. Frozen red cells revisited. N.Engl.J.Med 1984;311(26):1696-8. 17. Crowley JP, Wade PH, Wish C, Valeri CR. The purification of red cells for transfusion
by freeze-preservation and washing. V. Red cell recovery and residual leukocytes after freeze-preservation with high concentrations of glycerol and washing in various systems. Transfusion 1977;17(1):1-7.
18. Chaplin HJ. The proper use of previously frozen red blood cells for transfusion. Blood 1982;59(6):1118-20.
Chapter 2 Stability after thawing of RBCs frozen with the high- and low-glycerol method
19. Huggins C. Preparation and usefulness of frozen blood. Annu.Rev.Med 1985;36:499-503.
20. Valeri CR. Frozen blood (concluded). N.Engl.J.Med. 1966;275(8):425-31.
21. Standards for Blood Banks and Transfusion Services, 20th edition, Bethesda: AABB, 2000.
22. Guide to the Preparation, Use and Quality Assurance of Blood Components, 7th edition, Strasbourg: Council of Europe, 2001
23. Hess JR, Hill HR, Oliver CK, Lippert LE, Greenwalt TJ. The effect of two additive solutions on the postthaw storage of RBCs. Transfusion 2001;41(7):923-7. 24. Valeri CR, Ragno G, Pivacek LE, Srey R, Hess JR, Lippert LE, Mettille F, Fahie
R, O’Neill EM, Szymanski IO. A multicenter study of in vitro and in vivo values in human RBCs frozen with 40-percent (wt/vol) glycerol and stored after deglycerolization for 15 days at 4 degrees C in AS-3: assessment of RBC processing in the ACP 215. Transfusion 2001;41(7):933-9.
25. Valeri CR, Ragno G, Pivacek L, O’Neill EM. In vivo survival of apheresis RBCs, frozen with 40-percent (wt/vol) glycerol, deglycerolized in the ACP 215, and stored at 4 degrees C in AS-3 for up to 21 days. Transfusion 2001;41(7):928-32. 26. Crowley JP, Skrabut EM, Valeri CR. Immunocompetent lymphocytes in previously
frozen washed red cells. Vox Sang. 1974;26(6):513-7.
27. Crowley JP, Valeri CR. The purification of red cells of transfusion by freeze preservation and washing. II. The residual leukocytes, platelets, and plasma in washed,freeze-preserved red cells. Transfusion 1974;14(3):196-202.
28. Krijnen HW, Kuivenhoven AC, De Wit JJ. The preservation of blood cells in the frozen state. Experiences and current methods in the Netherlands. Cryobiology 1968;5(2):136-43.
29. Zijlstra WG, Van Kampen E. Standardization of hemoglobinometry. I. The extinction coefficient of hemiglobincyanide. Clin.Chim.Acta 1960;5:719-26. 30. Vogelaar EF, Brummelhuis HG, Beentjes SP, Krijnen HW. Contributions to the
optimal use of human blood. I. Analysis and optimalization of the production of plasma protein fraction (PPF). Vox Sang. 1972;23(6):481-92.
31. Lutz HU, Stammler P, Fasler S, Ingold M, Fehr J. Density separation of human red blood cells on self forming Percoll gradients: correlation with cell age. Biochim. Biophys.Acta 1992;1116(1):1-10.
32. de Korte D, Haverkort WA, Roos D, van Gennip AH. Anion-exchange high performance liquid chromatography method for the quantitation of nucleotides in human blood cells. Clin.Chim.Acta 1985;148(3):185-96.
33. de Korte D, Haverkort WA, van Gennip AH, Roos D. Nucleotide profiles of normal human blood cells determined by high-performance liquid chromatography. Anal. Biochem. 1985;147(1):197-209.
34. Hogman CF, de Verdier CH, Ericson A, Hedlund K, Sandhagen B. Studies on the mechanism of human red cell loss of viability during storage at +4 degrees C in vitro. I. Cell shape and total adenylate concentration as determinant factors for posttransfusion survival. Vox Sang. 1985;48(5):257-68.
35. Valeri CR, Pivacek LE, Cassidy GP, Ragno G. Posttransfusion survival (24-hour) and hemolysis of previously frozen, deglycerolized RBCs after storage at 4 degrees C for up to 14 days in sodium chloride alone or sodium chloride supplemented with additive solutions. Transfusion 2000;40(11):1337-40.
36. Valeri CR, Pivacek LE, Cassidy GP, Ragno G. The survival, function, and hemolysis of human RBCs stored at 4 degrees C in additive solution (AS-1, AS-3, or AS-5) for 42 days and then biochemically modified, frozen, thawed, washed, and stored at 4 degrees C in sodium chloride and glucose solution for 24 hours. Transfusion 2000;40(11):1341-5.
37. Moore GL, Ledford ME, Mathewson PJ, Hankins DJ, Shah SB. Post-thaw storage at 4 degrees C of previously frozen red cells with retention of 2,3-DPG. Vox Sang. 1987;53(1):15-8.
38. Derrick JB, McConn R, Sorovacu ML, Rowe AW. Studies of the metabolic integrity of human red blood cells after cryopreservation. II. Effects of low-glycerol-rapid-freeze preservation on glycolysis. Transfusion 1972;12(6):400-4.
39. Noorman F, Lelkens CCM, et al. Frozen red blood cells, comparison of the low glycerol and high glycerol method. Transfusion 2000;40:(Suppl):63S.
40. Rittmeyer IC, Nydegger UE. Influence of the cryoprotective agents glycerol and hydroxyethyl starch on red blood cell ATP and 2,3-diphosphoglyceric acid levels. Vox Sang. 1992;62(3):141-5.
41. Pietersz RN, de Korte D, Reesink HW, Dekker WJ, van den Ende A, LOOS JA. Storage of whole blood for up to 24 hours at ambient temperature prior to component preparation. Vox Sang. 1989;56(3):145-50.
42. Moroff G, Meryman HT. Influence of glygerol on ATP and 2,3-DPG levels of human erythrocytes. Vox Sang. 1979;36(4):244-51.
Charles C.M. Lelkens, Jack G. Koning, Bob de Kort, Ingeborg B.G. Floot and Femke Noorman
Military Blood Bank, Leiden, the Netherlands
Transfus Apher Sci. 2006 Jun; 34(3):289-98
