What can we learn from cryopreserved blood cells?

Transcription

1 What can we learn from cryopreserved blood cells? Andreas Sputtek Universitätsklinikum Hamburg-Eppendorf Inst. f. Transfusionsmedizin Hamburg, Germany

2 Plenary Lecture (Definition) plenary* ) : full; complete; absolute lecture** ) : an informative talk given as before an audience and usually prepared beforehand * = This lecture is very subjective. ** = This lecture was completed last night.

3 Lesson 1 a) Never trust what is said in a Plenary Lecture. b) Doubt is one of the major driving forces to obtain scientific truth.

4 Hematopoiesis Bone marrow Myeloic progenitor Multipotential hematopoietic stem cell Lymphoid progenitor Megakaryocyte Erythroblast Myeloic precursor cell pre T-cell pre B-cell Peripheral blood Thrombocytes Erythrocytes Granulocytes Monocytes T-Lymphocytes B-Lymphocytes

5 Lesson 2 Peripheral blood cells are an ideal study material because they are easily accessible.

6 Venipuncture: Peripheral Blood

7 N. Kröger 2005

8 Lesson 3 a) Bone marrow stem cells are not easily accessible. b) But peripheral blood stems (when accessable by apheresis) are more easily available.

9 Red blood cell (RBC) = Erythrocyte White blood cell (WBC) = Leukocyte Platelet (PLT) = Thrombocyte

10 Discovery of the Cryoprotectant Glycerol 1950 Smith A. U. Prevention of haemolysis during freezing and thawing of red blood-cells Lancet 259, (1950) ("After freezing quickly to, and storage at, -79 C for... 3 months, and subsequent thawing at +40 C, most of the red blood corpuscles survived and were unaltered in shape.")

11 Lesson 4 a) In the beginning of our discipline it was sufficient to describe a cooling rate as quick. b) It was also sufficient to give the temperature of the water bath used for thawing, and no specification of the warming rate was needed. c) Audrey Smith was British, because of the spelling of the word h(a)emolysis

12 Cooling Rate Definition T 1 temperature T 2 B = T 1 T 2 t 2 t 1 time t 1 t 2

13 Lesson 5 Cooling and warming rates specified in different publications may have the same numerical value, but the underlying temperature time-histories may be totally different.

14 Discovery of the Cryoprotectant Glycerol 1951 Sloviter H. A. Recovery of human red blood-cells after freezing Lancet 261, (1951) ("The slow removal of glycerol from red blood-cells has been successfully accomplished by dialysis against saline solutions containing progressively decreasing concentrations of glycerol.")

15 Lesson 6 K. Knickerbocker Osmotic phenomena during the removal of low molecular weight cryoprotectants (e.g. glycerol, Me 2 SO) after thawing are extremely relevant.

16 First Clinical Application of Frozen/Thawed Red Blood Cells April 16, 1951 Mollison P. L., Sloviter H. A. Hammersmith Hospital London Successful transfusion of previously frozen human red cells Lancet 261, (1951) ("... one patient severely ill with chronic leukaemia, who had already received many transfusions, was transfused with approximately 100 ml. of a suspension of previously frozen red cells. No unfavourable effects were observed and differential agglutination tests showed that the circulation contained approximately the expected number of red cells.")

17 Lesson 7 a) It was much easier to perform clinical studies in the beginning of our discipline than it is nowadays. b) Progress has slowed down because of an overregulation of clinical studies for saftety reasons. c) Nowadays pharmaceutical companies strongly influence the subject of research, because they have the money to pay for clinical studies.

18 Scaling up plasma buffy coat (WBC, PLT) RBC

19 Lession 8 In the old days no leukodepletion took place. The results were obtained with either partially buffy-coat removed RBC (at best), or no removal took place at all. Nowadays a WBC depleted RBC unit contains less than 1 million WBC, that corresponds to a WBC reduction by 3 to 4 orders of magnitude.

20 Red Cell Preservation Procedures High glycerol slow-cooling techniques 1960 Tullis Procedure, 1972 modified by Meryman! final glycerol concentration 40% to 50%! slow cooling (about 1 C/min) to 80 C! storage temperature: -80 C! continuous flow - centrifuge required for adding the glycerol prior to freezing and to remove it after thawing, in Meryman s method only standard blood bank equipment is needed! dominant method in the US Tullis J. L., Haynes L., Pyle H., Wallach S., Pennell R., Sproul, M. Khoubesserian A. Clinical use of frozen blood, Arch. Surg. 1, 169 (1960)

21 Red Cell Preservation Procedures High-glycerol slow-cooling techniques 1963 Huggins Procedure! original cryoprotectant dimethyl sulfoxide, later on abandoned in favor of glycerol! reversible agglomeration of red cells in a nonionic medium used for the removal of the cryoprotectant! clinical experience in combat casualties Huggins H. E. Preservation of blood for transfusion with dimethyl sulfoxide and a novel washing technique, Surgery 54, 191 (1963)

22 Red Cell Preservation Processes Low-glycerol rapid-cooling techniques Krijnen and Rowe Procedures!!!!! red cell recovery depends on both additive concentration and heat transfer during cooling final glycerol concentration 14% to 18% for rapid cooling/storage liquid nitrogen required only standard blood bank equipment needed for the introduction and removal of the cryoprotectant dominant method in Europe Krijnen, H. W., Kuivenhoven, A. C. J., De Wit, J. J. F. M. The preservation of blood cells in the frozen state. Experiences and current methods in the Netherlands, Cryobiology 5, (1968) Rowe, A. W., Eyster, E., Kellner, A. Liquid nitrogen preservation of red blood cells for transfusion: a low glycerol-rapid freeze procedure, Cryobiology 5, (1968)

23 Lession 9 Sometimes, when time has come, several people may have the same idea.

24 Advantages of Cryopreserved Red Cells " Reducing the infectious risks of homologous blood (Quarantine) " Extension of preoperative collection period for autologous blood prior to elective surgery " Date of surgery being not restricted by outdating of autologous units " Unlimited preservation without subsequent loss in quality (autologous deposits; inappropriate relief planning; temporary shortages) " Reducing problems regarding compatible blood (rare blood groups; multiple antibodies)

25 Lesson 10 a) At present all these advantages are not considered to outweigh the additional cost associated with cryopreservation. b) WBC removal (which used to be the dominant reason for cryopreservation in the past) can be achieved nowadays much better by a filtration process. c) Today homologous blood is safe because of extensive testing. This may change when a new pathogen shows up which cannot be detected by existing tests.

26 Osmotic Equilibrium electrolytes H 2 O osmotically inactive residual cell volume electrolytes H 2 O

27 Osmotic Dehydration electrolytes H 2 O osmotically inactive residual cell volume electrolytes electrolytes ice H 2 O electrolytes

28 Osmotic Shrinkage 100% volume lymphocytes critical volume Scheiwe & Körber % freezing temperature critical temperature osmotic inactive volume temperature

29 0 Water rich part of the phase diagram NaCl H 2 O -5 solution -10 T [ C] ice + solution T eut = C c = 22.4 % eut Data from: Landolt/Bernstein 1976 ice + NaCl. 2H 2O cnacl [%, w/w]

30 100 c [%, w/w] NaCl temperature hemolysis [%] concentration 20 RBC 0-2 Lovelock T [ C]

31 Lesson 11 a) RBC understand the liquidus curve of the phase diagram. c(free hemoglobin) b) hemolysis [%] = x (100 hematocrit [%]) c(total hemoglobin) In one of the most frequently cited papers regarding the introduction of trehalose into RBC prior to freezing the hematocrit was not considered, and that is not the only paper published where such a mistake has happened.

32 Intracellular Ice Formation electrolytes osmotically inactive residual cell volume electrolytes electrolytes H 2 O ice

33 lymphocytes Englich & Körber 1986

34 Two Factor Hypothesis 1 intracellular ice formation osmotic dehydration survival survival 0 cooling rate Mazur 1977

35 granulocytes (PMNC)? 1 intracellular ice formation osmotic dehydration survival 0 no survival cooling rate

36 100 yeast RBC Mazur & Schmidt 1968 Scheiwe & Nick recovery [%] 1 mouse BM marrow stem cells Leibo et al cooling rate [C /min]

37 Lesson 12 Diagrams showing data regarding fundamental aspects of cryobiology are subject to microbiological contamination.

38 M glycerol mouse BM stem cells 60 Recovery CFU spleen (%) M glycerol 0.4 M glycerol 0 M glycerol cooling rate (K/min) Leibo et al. 1970

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40 Lesson 13 a) Hydroxyethyl starch is the collective name for a group of modified starches. b) They are based on starch (mostly the amylopectin part of it) which has been modified by the substitution of a varying amount of hydroxyethyl groups per anhydroglucoes unit (DS, typically ).

41 CH 2 OR OR CH 2 OR OR OR OR OR R = H, CH2-CH2-OH OR n

42 Lesson 13 (continued) a) This polymer also varies largely with regard to the molecular weight average (M w, typically 70, ,000 g/mol) and the molecular weight distribution. b) So the term HES is as informative as the term sugar. c) For more details see: HES A Nightmare

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44 Rule of thumb: approx. 0.5 g H 2 O do not crystallize per 1 g of HES Körber & Scheiwe 1980

45 Van t Hoff s Law π V = n R T π = n/v R T c = n/v π = osmotic pressure π = c R T V = volume n = moles π/c = R T R = ideal gas constant T = temperature π/c = const. c = concentration

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47 [wt./wt.]

48 Lession 14 It is not true that such properties as osmotic pressure, freezing point depression, and vapor pressure depression of HES (and probably other polymers) in concentrations used for the cryopreservation of biological cells are strictly colligative properties (i.e. only depending on the number of molecules, not on their chemical properties).

49 Hct = 42% c(hes) = 11.5 wt.% RBC 60 recovery [%] B = 13 K/min n = c NaCl (EC) [mmol/l]

50 RBC 60 recovery [%] Hct = 42% c(hes) = 12 wt.% B = 70 K/min n = 4 c NaCl (EC) [mmol/l]

51 RBC recovery [%] Hct = 42% c(hes) = 12 wt.% B = 220 K/min n = 4 c NaCl (EC) [mmol/l]

52 RBC 60 recovery [%] Hct = 42% c(hes) = 12 wt.% B = 400 K/min n = 4 c NaCl (EC) [mmol/l]

53 Lession 15 (from a clinical phase 2 trial) a) The transfusion of 1 autologous unit (approx. 450 ml) of HES cryopreserved RBC is safe. b) A post-thaw washing step for the reduction of free hemoglobin and removal of HES is not needed for 1 unit but can be performed quickly in the case of the transfusion of more units. c) Further studies are needed in the case of the transfusion of larger volumes and homologous units. d) No sponsor found so far for a phase 3 trial (see Lesson 7).

54 Lession 16 There is never enough time in Plenary Lectures to cover the entire topic. Those interested in more details regarding the freezing of monocytes, platelets and peripheral hematopoietic stem cells are asked to contact the presenter for recent reviews on these topics: