CRYOBIOLOGY
13, l-8
(1976)
Cryopreservation
of Human
A Glycerol-Glucose, GEORGE
Moderate
DAYIAN
AND
Platelets
for Transfusion
Rate Cooling ARTHUR
Procedure 1
W. ROWE
The Cryobiology Laboratory of The Lindsley F. Kimball Research Institute, The New York Blood Center, 310 East 67th Street, New York, New York 10021
The growing need for human pIatelets in clinical medicine and the relatively short period during which they can be stored emphasize the potential usefulness of a method for their cryopreservation. The increased demand for platelets is illustrated by a steady increase in the number of units prepared at The New York BIood Center, from 56,370 in 1970 to 105,200 in 1974. A number of investigators have advocated the use of dimethyl sulfoxide (DMSO) as a cryopreservative agent for platelets (15, 16, 18, 21, 23). However, the toxicity and odor associated with this agent preclude its use unless the thawed platelets are washed to remove the DMSO prior to transfusion. In this report a method for platelet cryopreservation is described which makes use of a glycerol-glucose mixture as the cryopreservative agent which permits the transfusion of the thawed platelets without prior washing to remove the additive. In &ro assays of the frozen-thawed platelets indicate that they compare favorably with fresh platelets when measured by these criteria. Received September 30, 1975. 1 A preliminary report has been presented at the annual meetings of the Society for Cryobiology, August 1974, in London (14) and the American Association of Blood Banks, November 1974, in Anaheim, California ( 13). This research was supported by an NHLI grant, No. HL-99011, and by a grant from The Union Carbide Corporation.
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Processing of Platelets for Freezing Human platelet-rich plasma (PRP) was prepared from blood drawn in acid-citratedextrose (ACD, NIH Formula A) and its processing started within 3 hr after donation using unchilled PRP: (i) A volume of ACD equal to 10% of the weight of PRP was injected into the bag in order to Iower the pH to about 6.5 and prevent aggregation. The mixture was centrifuged at 4000 rpm for 5 min in a Sorvall RC-3 centrifuge fitted with an HGL-4 head. The platelet-poor plasma (PPP) was separated by use of a Fenwal plasma extractor and retained for later use in preparation of the freezing solution and for resuspension of thawed platelets. (ii) The platelet concentrate (PC) was resuspended in a minimal quantity of its residual plasma (approximately 10 ml) by gentIe kneading of the bag and 300 ml of the freezing solution added. The Iatter consisted of a mixture of 90 ml of autologous PPP and 210 ml of a solution containing 7.14% (v/v) glycerol, 5.71% (w/v) glucose and 0.9% (w/v) NaCI. The final concentration of glycerol was 5% and that of glucose was 4%. (iii) The suspension of glycerolized platelets was centrifuged, the supernatant fluid removed, and the PC resuspended in some residua1 freezing solution (approximately 4-5 ml per unit)
as before.
(iv)
Air was
DAYIAN
AND
TABLE Recovery Experiment
1A
Platelets
from Platelet-Rich
Fresh platelets 7”:m
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
of Frozen-Thawed
ROWE
248 261 234 210 169 204 189 211 221 249 204 253 188 207 195 227 184 224 209 227 237 204 281 288 315 289
Plasmaa Frozen-thawed
:~%y 6
PRP total (X10”)
5.26 3.73 4.67 6.70 6.05 5.52 4.47 5.21 4.47 5.72 6.20 6.92 4.27 4.39 5.84 4.06 4.64 4.90 6.61 4.75 4.10 5.04 3.89 5.52 4.45 3.23
1.30 0.974 1.09 1.41 1.02 1.13 0.845 1.10 0.988 1.42 1.26 1.75 0.803 0.909 1.14 0.922 0.854 1.10 1.38 1.08 0.972 1.03 1.09 1.59 1.40 0.933
PC/J6 (X10’)
1.21 1.22 0.935 1.05 1.62 1.65 1.29 0.825 0.785 1.90 1.80 1.93 1.01 0.830 0.990 1.09 1.37 1.63 2.07 1.84 1.38 1.47 1.32 1.70 1.21 1.28
platelets
PC total (X10”)
R”c?T”0
0.687 0.695 0.533 0.598 0.923 0.938 0.735 0.470 0.450 1.08 1.02 1.10 0.570 0.690 0.560 0.630 0.781 0.930 1.18 1.05 0.790 0.840 0.750 0.986 0.700 0.730
53 71 49 42 90 83 87 43 46 ‘76 81 63 71 76 49 68 91 85 86 97 81 82 69 62 50 78
Average
70
a Abbreviations: PRP, platelet-rich plasma; PC, platelet concentrate. b The volume of PC varied from 44 to 60 ml, with an average of 57 ml. c Recovery calculations reflect losses both in the processing and freeze-thawing
injected into the bag and the PC removed into a syringe. The PC remaining in the bag was removed by two washings with 1.5 ml each of freezing solution supernatant. The PC (approximately 7 ml per unit) was injected into a blood-freezing bag (UCAR Style 0750-2, Union Carbide)) the air removed and the bag heat-sealed. Freezing and Thawing
of Platelets
The seaIed bag was placed between two aluminum cover plates, 0.8 mm thick, and clamped. The bag containing the platelets to be frozen, together with a similar bag containing the freezing solution without
procedures.
platelets, but fitted with a copper-constantan thermocouple, were placed on a stand in the Linde biological freezer (Union Carbide) so that the bags did not touch each other. A controlled rate of cooling was achieved by use of the override switch on the Linde biological freezer controller and observation of the recorded temperature. Continuous temperature readings were recorded on the Honeywell sectronic 18. The rate of cooling was not greater than 30°C per min until the heat of fusion produced a plateau in the curve (approximately -7°C). A similar rate of 30°C per
CRYOPRESERVATION
OF PLATELETS TABLE
IN GLYCEROGGLUCOSE
3
1B
Recovery of Frozen-Thawed Platelets by an Independent Laboratorya Experiment
Fresh platelets PRPb (ml)
1 2 3 4 5 6 7 8 9 10
Frozen-thawed PRP total (X IO”)
PC/b@ (X 106)
2.27 2.15
1.72 3.01 3.33
209
8.96 7.98 7.98 10.6
228 240
7.55
1.72
5.95
1.43 1.74 3.05 1.83 1.35
253 270
259 248 288 250
“;“,‘;g
6.72 12.3 6.34 5.38
1.90 2.22
Average
2.94 1.79 1.89 1.89 4.61 2.43
1.84
platelets
;“x ;$y
Reczw’D
0.960
42
1.70 1.86 1.38
79
0.789 1.10 1.14 2.67
1.36 1.04
98 62 46 77 66 88 74 77 71
0 We are indebted to Dr. M. Stryker and J. van der Sande of The Blood Derivatives Laboratory of The New York Blood Center for carrying out these determinations. b Abbreviations: PRP, platelet-rich plasma; PC, platelet concentrate. c Recovery calcuIatious reflect losses both in t,he processing and freeze-thawing procedures.
min was continued until the temperature reached -8O”C, at which point the freezing assembly was transferred into liquid nitrogen for storage. The frozen platelets were thawed by submersion of the assembly in a 40°C water bath with mild agitation for 20 sec. If some aggregation occurred, the platelets were kneaded by hand until they dispersed. The thawed platelets were immediately reconstituted in 50 ml of autologous platelet-free plasma (PFP) and incubated at room temperature until “swirling” was established, an average of l-2 hr.
In Vitro Assay of PZateZets Plutelet recovery. The yield of platelets was determined by counting the platelets in a hemocytometer chamber by phase contrast microscopy, according to the method of Brecher and Cronkite (6). Size distribution. Platelet size distribution patterns were obtained by use of a Coulter counter, Model ZBI, a size distribution analyzer, Model P64, and x-y recorder II (20). The 70-c” aperture electrode was used with the following counter settings: I/amplification = 4, l/aperture
current = 0.177, matching switch = 2OK, and gain trim = 2.5. Window settings varied from a lower threshold of 8 to an upper threshoId of 100. The count range setting on the analyzer was 4096. Latex particles (Coulter), 2.02 pm in diameter, were used to calibrate the instrument. Platelets were resuspended in Isoton (Coulter) to give a concentration of about 4OO,OOO/mI. Serotonin uptake. The uptake of [W] scrotonin by platelets was studied by the method of Born and Gillson (3). [3-W] hydroxytryptamine creatinine sulfate (55 mCi/mmol, Amersham) was dissolved in 70% alcohol to give a final concentration of 10 &i/ml and stored at -20°C (17). To 4 ml of platelet suspension was added 0.01 ml of the [14C]serotonin and the mixture incubated at 37°C for 30 min. Radioactivity was determined on O.l-ml samples of platelet suspensions as well as on platelet-free aliquots after centrifugation. Aggregation. Adenosine diphosphate ( ADP)-induced aggregation was performed turbidometrically with a Chronolog Aggregometer (4,5). Thawed reconstituted platelets were incubated with 1 mg/ml of
DAYIAN
AND ROWE
A
CRYOPRESERVATION
OF PLATELETS
the enzyme, apyrase (Sigma) for I hr at 37°C (1). This treatment restored the sensitivity of the platelets refractory to ADP-induced aggregation. The mixture was centrifuged at 4500g and resuspended in nonacidified heterologous PFP with added calcium chloride (4 mM). Aggregation was induced by addition of 200 $4 ADP to a platelet suspension of 240,000/mm3. Clot retraction. Clot retraction induced by the addition of thrombin was performed according to the method of Bettex-Galland and Liischer (2) as described for PRP. Platelets were diluted to 180,000/mms in 5 ml of a mixture consisting of 60% plasma, 6 mM imidazole-HCl buffer, pH 7.4, 1 mM glucose and 60 mM NaCI. The suspension was preincubated for 5 min at 37°C before the addition of 0.5 ml of thrombin (6 units) in 2 mM CaC12. Incubation was continued at 37°C. RESULTS
Recove y of Platelets after Freeze-Thawing After overnight storage in liquid nitrogen, the platelets were thawed and counted by phase microscopy and compared with the numbers of platelets in the starting platelet-rich plasma. Table IA Iists the results for 26 preparations performed on a routine basis in this laboratory. Recoveries ranged from 42 to 97% with an average of 70%. Some 73% of the preparations gave recoveries greater than 60%. In order to determine whether the written protocol could be repeated independently by other workers, members of the staff of the BIood Derivatives Laboratory at The New York Blood Center kindly agreed to complete 10 preparations. These resuhs are listed in Table 1B. Recoveries ranged from 42 to 98% with an average of 71%, in confirmation of the results shown in Table IA.
IN GLYCEROGGLUCOSE
5
In a separate group of five experiments, the average loss of pIateIets was found to be 29%, of which 16% was accounted for in the “platelet-poor plasma” and 8% left in the freezing solution supernatant. The remainder (5%) was lost during transfer of platelets from the processing bag. These three areas of losses accounted for approximately 97% of the total loss. During these studies it was noted that aggregation of thawed or reconstituted platelets can be prevented or minimized by complete removal of erythrocytes in the PRP or PC. This can be accomplished by centrifugation of PRP or of PC, resuspended in freezing solution, at 800 rpm for 2-3 min in the SorvaII RC-3 centrifuge. Platelet Size Distribution Figure 1 illustrates the size distribution of platelets from fresh nonfrozen PRP (Fig. 1 A) compared with those of thawed reconstituted platelets (Fig. 1 B). A narrow peak that is seen only as a shoulder in fresh platelets appears in the thawed samples below values of 1.5 pm3. Broader and often less sharply defined secondary peaks, with mean platelet size of 4 pm3, coincide with the primary peak of fresh platelet preparations. The number of large platelets make up a lesser proportion of the total thawed platelet population than in fresh platelets. In Fig. 1 C is shown the effect of washing the thawed platelets, resulting in the removal of some particles below 1.5 pm3. The resulting platelets display a pattern similar to those in PRP {Fig. 1 A). Serotonin Uptake Table 2 illustrates the results of measurements of serotonin uptake by fresh and frozen-thawed platelets. These show serotonin uptake by frozen-thawed platelets to be some 90% of that obtained with fresh platelets.
FIG. 1. Size distribution of platelets: (A), fresh platelets; ( C), frozen-thawed platelets washed by one centrifugation.
(B),
frozen-thawed
platelets;
and
DAYIAN
AND
ROWE
ADP I Rp
Frozen. Fresh
thawed
platelets
platelets
F t L 0
1
2
4
3 TIME
5
mm.
FIG. 2. ADP-induced aggregation of platelets: PPP refers to the control, without platelets; PRP refers to light transmission in the presence of platelets; ADP was added as indicated and aggregation measured with time.
FIG. 3. Clot retraction of fresh and frozen-thawed platelets. one on the left contained fresh, nonfrozen (NF), platelets, the an equal number of frozen-thawed-reconstituted (F) platelets. intervak from addition of thrombin were 10, 20, 30 and 40 min, right is a control tube (C) without platelets incubated for 40 min.
For each pair of tubes, the tube on the right contained From Ieft to right the time respectively. At the extreme
CRYOPRESERVATION TABLE Serotonin
OF PLATELETS
2
Uptake” --
Platelet preparation
Platelets/mm~
“$,y 0
Fresh
500,000 f 105,000 (342,500-735,000)
81.3 f 5.8 (60.3-87.9)
Frozen-thawed
450,000 i 165,000 (238,000-82.5,OOO)
73.3 i 10.9 (43.7-85.2)
a The number of experiments from 24 to 27.
ADP-Induced
in each group varied
Aggregation
Thawed reconstituted platelets were compared with fresh platelets with respect to the induction of aggregation by ADP. The results are illustrated in Fig. 2 for a typical experiment. Clot Retraction Figure 3 illustrates an experiment which compares the effect of fresh platelets (left tube of each pair) and of frozen-thawed platelets (right tube of each pair) at various time intervals after thrombin addition. The concentration of platelets in each instance was the same. The effectiveness of frozen-thawed platelets in inducing clot retraction was similar to that of fresh platelets. DISCUSSION
Development of the present method for cryopreservation of platelets was based on studies of the effect of freezing on platelet lysosomes (8-10). The use of relatively low concentrations of glycerol, which does not need to be removed prior to transfusion, together with glucose (12, 11) and a moderate rate of cooling has yielded a method which furnishes a good average yield (70% ) of intact platelets whose metabolic integrity is virtually intact. The recovery of intact platelets, following the method described in this report, has been confirmed by an independent group of investigators at The New York Blood Center. Some 20% of the platelets
IN GLYCEROL-GLUCOSE
7
are lost on the average due to transfers from bag to bag prior to freezing, whereas the freezing procedure itself accounts for less than 10% of the overall losses. Functional integrity of the frozen-thawed platelets was confirmed by measurement of serotonin uptake, aggregation response to added ADP and the ability of the platelets to cause clot retraction. The need for the enzyme apyrase in the test for aggregation underlines the presence of ADP in frozenthawed platelet suspensions, contributed by damaged platelets and contaminating erythrocytes. Although the size distribution patterns of fresh and frozen-thawed-reconstituted platelets differ (Figs. 1A and B), the pattern of the latter becomes comparable to that of fresh platelets following removal of erythrocyte debris and platelet fragments. Glycerol, with the important advantage over DMSO of nontoxicity and transfusibility, is a potentially useful cryoprotective additive. Cohen and Gardner (7) reported that freezing of platelets in 12% glycerol at a slow rate of cooling resulted in a relatively low recovery and observed diEiculties in removal of glycerol prior to transfusion. We have noted (8, 9) that 12% glycerol causes extensive damage to plateIet lysosomes and this platelet injury is further aggravated by freezing. Such lysosomal damage was not observed with 5% glycerol. We have also noted (10) that glycerol does not readily permeate human platelets and that platelets shrink in volume in direct response to the hypertonicity of the glycerol suspension medium. Osmotic stress obtained on removal of glycerol is most severe at high concentrations and is presumably due to differences in permeability rates between the entry of water and the exit of glycerol from platelets. Since the platelets preserved by this method appear to have retained their integrity based on several in uitro criteria, this approach to freezing has an important
s
DAYIAN
clinical potential. Because the glycerol concentration is low, it is possible to transfuse the thawed platelets into recipients either directly without washing to remove the additive or after dilution with a small volume of plasma. Preliminary studies designed to measure the survival of platelets in humans indicate that such preserved cells are suitabIe for clinical use ( 13, 14). ACKNOWLEDGMENTS We wish to express our appreciation to Drs. A. Kellner and A. Mazur for their support, encouragement and editorial comments. We are also indebted to Sing Chin, Nathan Strick, Martin Cohen, and Leslie Lenny for their expert technical assistance and to Herman Keck for secretarial assistance. Appreciation is also expressed to Dr. M. Stryker and J. van der Sande of the Blood Derivatives Laboratory of The New York Blood Center for independently confirming the platelet recovery data. REFERENCES 1. Ardlie, N. G., Perry, D. W., Packham, M. A., and Mustard, J. F. Influence of apyrase on stability of suspensions of washed rabbit platelets. Proc. Sot. Erp. BioZ. Med. 136, 1021 ( 1971). 2. Bettex-Galland, M. In “Thrombosis and Bleeding Disorders. Theory and Methods.” (N. U. Bang, F. K. Belier, E. Deutsch, and E. F. Mammen, Eds.), p. 442. Academic Press, New York, 1971. 3. Born, G. V. F., and Gillson, R. E. Studies on the uptake of 5-hydroxytryptamine by blood platelets. J. Physiol. 146, 472 ( 1959). 4. Born, G. V. R. Aggregation of blood platelets and its reversal. Nature (London) 194, 927 ( 1962). 5. Born, G. V. R., and Cross, N. J. The aggregation of blood platelets. J. Physiol. 168, 178 (1963). 6. Brecher, G., and Cronkite, E. P. Morphology and enumeration of human blood platelets. j. Awl. Physiol. 3, 365 ( 1950). 7. Cohen, P., and Gardner, F. H. Platelet preservation. IV. Preservation of human platelet concentrates by controlled slow freezing in a glycerol medium. N. Eng. J. Med. 274, 1400-1407 ( 1966). 8. Dayian, G., and Rowe, A. W. Activation of lysosomal enzymes in human platelets during cryopreservation. Cryobiology 6, 579 ( 1970). 9. Dayian, G., Chin, S. N., and Rowe, A. W. Cryoprotection of human platelets: The
AND ROWE influence of cooling rate and glycerol concentration on activating the lysosomal enzyme, p-glucuronidase. Cr~obioIog~ 8, 393 (1971). 10. Dayian, G., Chin, S. N., and Rowe, A. W. Differences in platelet permeability to glycerol and DMSO. CryobioIogq 8, 376 (1971). 11. Dayian, G., Cohen, M., Chin, S. N., and Rowe, A. W. ADP-aggregation in platelets frozen with glycerol. CryobioZogy 9, 329 (1972). 12. Dayian, G., Chin, S. N., Cohen, M., and Rowe, A. W. Freezing of human platelets in a glycerol-glucose medium. Cryobiology 10, 526 ( 1973). 13. Dayian, G., Reich, L., Mayer, K., Turc, J. M., and Rowe, A. W. Transfusion of human platelets after freezing in a glycerol-glucose medium. Transfusion 14, 511 ( 1974). 14. Dayian, G., Reich, L., Mayer, K., Turc, J. M., and Rowe, A. W. Use of glycerol to preserve platelets suitable for transfusion. Cryobiology 11, 563 (1974). 15. Djerassi, I., Farber, S., Roy, A., and Cavins, J. Preparation and in u&o circulation of human platelets preserved with combined dimethyl sulfoxide and dextrose. Tmnsfusion 6, 572-576 ( 1966). 16. Handin, R. I., and Valeri, C. R. Improved viability of previously frozen platelets. Bbod 40, 509-513 ( 1972). 17. Jerushalmy, Z., and Zucker, M. B. Some effects of fibrinogen degradation products on blood platelets. Thomb. Diath. Haemorrhag. 15, 413 (1966). 18. Kim, B. K., and Baldini, M. G. Preservation of viable platelets by freezing. Effect of plastic containers. PYOC. SOC. Erp. Biol. Med. 142, 345 ( 1973). 19. Marcus, A. J., and Zucker, M. B. “The Physiology of Blood Platelets.” Grune and Stratton, New York, 1965. 20. McDonald, T. P., Odell, T. T., Jr., and Goslee, D. G. Platelet size distribution in relation to age. Proc. Sot. Exp. Biol. Med. 115, 684 (1964). 21. Murphy, S., Sayar, S. N., Abdou, N. L., and Gardner, F. H. Platelet preservation by freezing. Use of dimethyl sulfoxide as cryoprotective agent. Transfusion 14, 139144 ( 1974). 22. Rowe, A. W., and Peterson, J. Effect of glycerol, HES and DMSO on functional integrity of human blood platelets before and after freezing. Cryobiology 8, 393 ( 1971). 23. Valeri, C. R., Feingold, H., and Marchionni, L. D. A simple method for freezing human platelets using 6% dimethyl sulfoxide and storage at -80°C. Blood 43, 131-146 (1974).
13, l-8
(1976)
Cryopreservation
of Human
A Glycerol-Glucose, GEORGE
Moderate
DAYIAN
AND
Platelets
for Transfusion
Rate Cooling ARTHUR
Procedure 1
W. ROWE
The Cryobiology Laboratory of The Lindsley F. Kimball Research Institute, The New York Blood Center, 310 East 67th Street, New York, New York 10021
The growing need for human pIatelets in clinical medicine and the relatively short period during which they can be stored emphasize the potential usefulness of a method for their cryopreservation. The increased demand for platelets is illustrated by a steady increase in the number of units prepared at The New York BIood Center, from 56,370 in 1970 to 105,200 in 1974. A number of investigators have advocated the use of dimethyl sulfoxide (DMSO) as a cryopreservative agent for platelets (15, 16, 18, 21, 23). However, the toxicity and odor associated with this agent preclude its use unless the thawed platelets are washed to remove the DMSO prior to transfusion. In this report a method for platelet cryopreservation is described which makes use of a glycerol-glucose mixture as the cryopreservative agent which permits the transfusion of the thawed platelets without prior washing to remove the additive. In &ro assays of the frozen-thawed platelets indicate that they compare favorably with fresh platelets when measured by these criteria. Received September 30, 1975. 1 A preliminary report has been presented at the annual meetings of the Society for Cryobiology, August 1974, in London (14) and the American Association of Blood Banks, November 1974, in Anaheim, California ( 13). This research was supported by an NHLI grant, No. HL-99011, and by a grant from The Union Carbide Corporation.
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Processing of Platelets for Freezing Human platelet-rich plasma (PRP) was prepared from blood drawn in acid-citratedextrose (ACD, NIH Formula A) and its processing started within 3 hr after donation using unchilled PRP: (i) A volume of ACD equal to 10% of the weight of PRP was injected into the bag in order to Iower the pH to about 6.5 and prevent aggregation. The mixture was centrifuged at 4000 rpm for 5 min in a Sorvall RC-3 centrifuge fitted with an HGL-4 head. The platelet-poor plasma (PPP) was separated by use of a Fenwal plasma extractor and retained for later use in preparation of the freezing solution and for resuspension of thawed platelets. (ii) The platelet concentrate (PC) was resuspended in a minimal quantity of its residual plasma (approximately 10 ml) by gentIe kneading of the bag and 300 ml of the freezing solution added. The Iatter consisted of a mixture of 90 ml of autologous PPP and 210 ml of a solution containing 7.14% (v/v) glycerol, 5.71% (w/v) glucose and 0.9% (w/v) NaCI. The final concentration of glycerol was 5% and that of glucose was 4%. (iii) The suspension of glycerolized platelets was centrifuged, the supernatant fluid removed, and the PC resuspended in some residua1 freezing solution (approximately 4-5 ml per unit)
as before.
(iv)
Air was
DAYIAN
AND
TABLE Recovery Experiment
1A
Platelets
from Platelet-Rich
Fresh platelets 7”:m
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
of Frozen-Thawed
ROWE
248 261 234 210 169 204 189 211 221 249 204 253 188 207 195 227 184 224 209 227 237 204 281 288 315 289
Plasmaa Frozen-thawed
:~%y 6
PRP total (X10”)
5.26 3.73 4.67 6.70 6.05 5.52 4.47 5.21 4.47 5.72 6.20 6.92 4.27 4.39 5.84 4.06 4.64 4.90 6.61 4.75 4.10 5.04 3.89 5.52 4.45 3.23
1.30 0.974 1.09 1.41 1.02 1.13 0.845 1.10 0.988 1.42 1.26 1.75 0.803 0.909 1.14 0.922 0.854 1.10 1.38 1.08 0.972 1.03 1.09 1.59 1.40 0.933
PC/J6 (X10’)
1.21 1.22 0.935 1.05 1.62 1.65 1.29 0.825 0.785 1.90 1.80 1.93 1.01 0.830 0.990 1.09 1.37 1.63 2.07 1.84 1.38 1.47 1.32 1.70 1.21 1.28
platelets
PC total (X10”)
R”c?T”0
0.687 0.695 0.533 0.598 0.923 0.938 0.735 0.470 0.450 1.08 1.02 1.10 0.570 0.690 0.560 0.630 0.781 0.930 1.18 1.05 0.790 0.840 0.750 0.986 0.700 0.730
53 71 49 42 90 83 87 43 46 ‘76 81 63 71 76 49 68 91 85 86 97 81 82 69 62 50 78
Average
70
a Abbreviations: PRP, platelet-rich plasma; PC, platelet concentrate. b The volume of PC varied from 44 to 60 ml, with an average of 57 ml. c Recovery calculations reflect losses both in the processing and freeze-thawing
injected into the bag and the PC removed into a syringe. The PC remaining in the bag was removed by two washings with 1.5 ml each of freezing solution supernatant. The PC (approximately 7 ml per unit) was injected into a blood-freezing bag (UCAR Style 0750-2, Union Carbide)) the air removed and the bag heat-sealed. Freezing and Thawing
of Platelets
The seaIed bag was placed between two aluminum cover plates, 0.8 mm thick, and clamped. The bag containing the platelets to be frozen, together with a similar bag containing the freezing solution without
procedures.
platelets, but fitted with a copper-constantan thermocouple, were placed on a stand in the Linde biological freezer (Union Carbide) so that the bags did not touch each other. A controlled rate of cooling was achieved by use of the override switch on the Linde biological freezer controller and observation of the recorded temperature. Continuous temperature readings were recorded on the Honeywell sectronic 18. The rate of cooling was not greater than 30°C per min until the heat of fusion produced a plateau in the curve (approximately -7°C). A similar rate of 30°C per
CRYOPRESERVATION
OF PLATELETS TABLE
IN GLYCEROGGLUCOSE
3
1B
Recovery of Frozen-Thawed Platelets by an Independent Laboratorya Experiment
Fresh platelets PRPb (ml)
1 2 3 4 5 6 7 8 9 10
Frozen-thawed PRP total (X IO”)
PC/b@ (X 106)
2.27 2.15
1.72 3.01 3.33
209
8.96 7.98 7.98 10.6
228 240
7.55
1.72
5.95
1.43 1.74 3.05 1.83 1.35
253 270
259 248 288 250
“;“,‘;g
6.72 12.3 6.34 5.38
1.90 2.22
Average
2.94 1.79 1.89 1.89 4.61 2.43
1.84
platelets
;“x ;$y
Reczw’D
0.960
42
1.70 1.86 1.38
79
0.789 1.10 1.14 2.67
1.36 1.04
98 62 46 77 66 88 74 77 71
0 We are indebted to Dr. M. Stryker and J. van der Sande of The Blood Derivatives Laboratory of The New York Blood Center for carrying out these determinations. b Abbreviations: PRP, platelet-rich plasma; PC, platelet concentrate. c Recovery calcuIatious reflect losses both in t,he processing and freeze-thawing procedures.
min was continued until the temperature reached -8O”C, at which point the freezing assembly was transferred into liquid nitrogen for storage. The frozen platelets were thawed by submersion of the assembly in a 40°C water bath with mild agitation for 20 sec. If some aggregation occurred, the platelets were kneaded by hand until they dispersed. The thawed platelets were immediately reconstituted in 50 ml of autologous platelet-free plasma (PFP) and incubated at room temperature until “swirling” was established, an average of l-2 hr.
In Vitro Assay of PZateZets Plutelet recovery. The yield of platelets was determined by counting the platelets in a hemocytometer chamber by phase contrast microscopy, according to the method of Brecher and Cronkite (6). Size distribution. Platelet size distribution patterns were obtained by use of a Coulter counter, Model ZBI, a size distribution analyzer, Model P64, and x-y recorder II (20). The 70-c” aperture electrode was used with the following counter settings: I/amplification = 4, l/aperture
current = 0.177, matching switch = 2OK, and gain trim = 2.5. Window settings varied from a lower threshold of 8 to an upper threshoId of 100. The count range setting on the analyzer was 4096. Latex particles (Coulter), 2.02 pm in diameter, were used to calibrate the instrument. Platelets were resuspended in Isoton (Coulter) to give a concentration of about 4OO,OOO/mI. Serotonin uptake. The uptake of [W] scrotonin by platelets was studied by the method of Born and Gillson (3). [3-W] hydroxytryptamine creatinine sulfate (55 mCi/mmol, Amersham) was dissolved in 70% alcohol to give a final concentration of 10 &i/ml and stored at -20°C (17). To 4 ml of platelet suspension was added 0.01 ml of the [14C]serotonin and the mixture incubated at 37°C for 30 min. Radioactivity was determined on O.l-ml samples of platelet suspensions as well as on platelet-free aliquots after centrifugation. Aggregation. Adenosine diphosphate ( ADP)-induced aggregation was performed turbidometrically with a Chronolog Aggregometer (4,5). Thawed reconstituted platelets were incubated with 1 mg/ml of
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A
CRYOPRESERVATION
OF PLATELETS
the enzyme, apyrase (Sigma) for I hr at 37°C (1). This treatment restored the sensitivity of the platelets refractory to ADP-induced aggregation. The mixture was centrifuged at 4500g and resuspended in nonacidified heterologous PFP with added calcium chloride (4 mM). Aggregation was induced by addition of 200 $4 ADP to a platelet suspension of 240,000/mm3. Clot retraction. Clot retraction induced by the addition of thrombin was performed according to the method of Bettex-Galland and Liischer (2) as described for PRP. Platelets were diluted to 180,000/mms in 5 ml of a mixture consisting of 60% plasma, 6 mM imidazole-HCl buffer, pH 7.4, 1 mM glucose and 60 mM NaCI. The suspension was preincubated for 5 min at 37°C before the addition of 0.5 ml of thrombin (6 units) in 2 mM CaC12. Incubation was continued at 37°C. RESULTS
Recove y of Platelets after Freeze-Thawing After overnight storage in liquid nitrogen, the platelets were thawed and counted by phase microscopy and compared with the numbers of platelets in the starting platelet-rich plasma. Table IA Iists the results for 26 preparations performed on a routine basis in this laboratory. Recoveries ranged from 42 to 97% with an average of 70%. Some 73% of the preparations gave recoveries greater than 60%. In order to determine whether the written protocol could be repeated independently by other workers, members of the staff of the BIood Derivatives Laboratory at The New York Blood Center kindly agreed to complete 10 preparations. These resuhs are listed in Table 1B. Recoveries ranged from 42 to 98% with an average of 71%, in confirmation of the results shown in Table IA.
IN GLYCEROGGLUCOSE
5
In a separate group of five experiments, the average loss of pIateIets was found to be 29%, of which 16% was accounted for in the “platelet-poor plasma” and 8% left in the freezing solution supernatant. The remainder (5%) was lost during transfer of platelets from the processing bag. These three areas of losses accounted for approximately 97% of the total loss. During these studies it was noted that aggregation of thawed or reconstituted platelets can be prevented or minimized by complete removal of erythrocytes in the PRP or PC. This can be accomplished by centrifugation of PRP or of PC, resuspended in freezing solution, at 800 rpm for 2-3 min in the SorvaII RC-3 centrifuge. Platelet Size Distribution Figure 1 illustrates the size distribution of platelets from fresh nonfrozen PRP (Fig. 1 A) compared with those of thawed reconstituted platelets (Fig. 1 B). A narrow peak that is seen only as a shoulder in fresh platelets appears in the thawed samples below values of 1.5 pm3. Broader and often less sharply defined secondary peaks, with mean platelet size of 4 pm3, coincide with the primary peak of fresh platelet preparations. The number of large platelets make up a lesser proportion of the total thawed platelet population than in fresh platelets. In Fig. 1 C is shown the effect of washing the thawed platelets, resulting in the removal of some particles below 1.5 pm3. The resulting platelets display a pattern similar to those in PRP {Fig. 1 A). Serotonin Uptake Table 2 illustrates the results of measurements of serotonin uptake by fresh and frozen-thawed platelets. These show serotonin uptake by frozen-thawed platelets to be some 90% of that obtained with fresh platelets.
FIG. 1. Size distribution of platelets: (A), fresh platelets; ( C), frozen-thawed platelets washed by one centrifugation.
(B),
frozen-thawed
platelets;
and
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ADP I Rp
Frozen. Fresh
thawed
platelets
platelets
F t L 0
1
2
4
3 TIME
5
mm.
FIG. 2. ADP-induced aggregation of platelets: PPP refers to the control, without platelets; PRP refers to light transmission in the presence of platelets; ADP was added as indicated and aggregation measured with time.
FIG. 3. Clot retraction of fresh and frozen-thawed platelets. one on the left contained fresh, nonfrozen (NF), platelets, the an equal number of frozen-thawed-reconstituted (F) platelets. intervak from addition of thrombin were 10, 20, 30 and 40 min, right is a control tube (C) without platelets incubated for 40 min.
For each pair of tubes, the tube on the right contained From Ieft to right the time respectively. At the extreme
CRYOPRESERVATION TABLE Serotonin
OF PLATELETS
2
Uptake” --
Platelet preparation
Platelets/mm~
“$,y 0
Fresh
500,000 f 105,000 (342,500-735,000)
81.3 f 5.8 (60.3-87.9)
Frozen-thawed
450,000 i 165,000 (238,000-82.5,OOO)
73.3 i 10.9 (43.7-85.2)
a The number of experiments from 24 to 27.
ADP-Induced
in each group varied
Aggregation
Thawed reconstituted platelets were compared with fresh platelets with respect to the induction of aggregation by ADP. The results are illustrated in Fig. 2 for a typical experiment. Clot Retraction Figure 3 illustrates an experiment which compares the effect of fresh platelets (left tube of each pair) and of frozen-thawed platelets (right tube of each pair) at various time intervals after thrombin addition. The concentration of platelets in each instance was the same. The effectiveness of frozen-thawed platelets in inducing clot retraction was similar to that of fresh platelets. DISCUSSION
Development of the present method for cryopreservation of platelets was based on studies of the effect of freezing on platelet lysosomes (8-10). The use of relatively low concentrations of glycerol, which does not need to be removed prior to transfusion, together with glucose (12, 11) and a moderate rate of cooling has yielded a method which furnishes a good average yield (70% ) of intact platelets whose metabolic integrity is virtually intact. The recovery of intact platelets, following the method described in this report, has been confirmed by an independent group of investigators at The New York Blood Center. Some 20% of the platelets
IN GLYCEROL-GLUCOSE
7
are lost on the average due to transfers from bag to bag prior to freezing, whereas the freezing procedure itself accounts for less than 10% of the overall losses. Functional integrity of the frozen-thawed platelets was confirmed by measurement of serotonin uptake, aggregation response to added ADP and the ability of the platelets to cause clot retraction. The need for the enzyme apyrase in the test for aggregation underlines the presence of ADP in frozenthawed platelet suspensions, contributed by damaged platelets and contaminating erythrocytes. Although the size distribution patterns of fresh and frozen-thawed-reconstituted platelets differ (Figs. 1A and B), the pattern of the latter becomes comparable to that of fresh platelets following removal of erythrocyte debris and platelet fragments. Glycerol, with the important advantage over DMSO of nontoxicity and transfusibility, is a potentially useful cryoprotective additive. Cohen and Gardner (7) reported that freezing of platelets in 12% glycerol at a slow rate of cooling resulted in a relatively low recovery and observed diEiculties in removal of glycerol prior to transfusion. We have noted (8, 9) that 12% glycerol causes extensive damage to plateIet lysosomes and this platelet injury is further aggravated by freezing. Such lysosomal damage was not observed with 5% glycerol. We have also noted (10) that glycerol does not readily permeate human platelets and that platelets shrink in volume in direct response to the hypertonicity of the glycerol suspension medium. Osmotic stress obtained on removal of glycerol is most severe at high concentrations and is presumably due to differences in permeability rates between the entry of water and the exit of glycerol from platelets. Since the platelets preserved by this method appear to have retained their integrity based on several in uitro criteria, this approach to freezing has an important
s
DAYIAN
clinical potential. Because the glycerol concentration is low, it is possible to transfuse the thawed platelets into recipients either directly without washing to remove the additive or after dilution with a small volume of plasma. Preliminary studies designed to measure the survival of platelets in humans indicate that such preserved cells are suitabIe for clinical use ( 13, 14). ACKNOWLEDGMENTS We wish to express our appreciation to Drs. A. Kellner and A. Mazur for their support, encouragement and editorial comments. We are also indebted to Sing Chin, Nathan Strick, Martin Cohen, and Leslie Lenny for their expert technical assistance and to Herman Keck for secretarial assistance. Appreciation is also expressed to Dr. M. Stryker and J. van der Sande of the Blood Derivatives Laboratory of The New York Blood Center for independently confirming the platelet recovery data. REFERENCES 1. Ardlie, N. G., Perry, D. W., Packham, M. A., and Mustard, J. F. Influence of apyrase on stability of suspensions of washed rabbit platelets. Proc. Sot. Erp. BioZ. Med. 136, 1021 ( 1971). 2. Bettex-Galland, M. In “Thrombosis and Bleeding Disorders. Theory and Methods.” (N. U. Bang, F. K. Belier, E. Deutsch, and E. F. Mammen, Eds.), p. 442. Academic Press, New York, 1971. 3. Born, G. V. F., and Gillson, R. E. Studies on the uptake of 5-hydroxytryptamine by blood platelets. J. Physiol. 146, 472 ( 1959). 4. Born, G. V. R. Aggregation of blood platelets and its reversal. Nature (London) 194, 927 ( 1962). 5. Born, G. V. R., and Cross, N. J. The aggregation of blood platelets. J. Physiol. 168, 178 (1963). 6. Brecher, G., and Cronkite, E. P. Morphology and enumeration of human blood platelets. j. Awl. Physiol. 3, 365 ( 1950). 7. Cohen, P., and Gardner, F. H. Platelet preservation. IV. Preservation of human platelet concentrates by controlled slow freezing in a glycerol medium. N. Eng. J. Med. 274, 1400-1407 ( 1966). 8. Dayian, G., and Rowe, A. W. Activation of lysosomal enzymes in human platelets during cryopreservation. Cryobiology 6, 579 ( 1970). 9. Dayian, G., Chin, S. N., and Rowe, A. W. Cryoprotection of human platelets: The
AND ROWE influence of cooling rate and glycerol concentration on activating the lysosomal enzyme, p-glucuronidase. Cr~obioIog~ 8, 393 (1971). 10. Dayian, G., Chin, S. N., and Rowe, A. W. Differences in platelet permeability to glycerol and DMSO. CryobioIogq 8, 376 (1971). 11. Dayian, G., Cohen, M., Chin, S. N., and Rowe, A. W. ADP-aggregation in platelets frozen with glycerol. CryobioZogy 9, 329 (1972). 12. Dayian, G., Chin, S. N., Cohen, M., and Rowe, A. W. Freezing of human platelets in a glycerol-glucose medium. Cryobiology 10, 526 ( 1973). 13. Dayian, G., Reich, L., Mayer, K., Turc, J. M., and Rowe, A. W. Transfusion of human platelets after freezing in a glycerol-glucose medium. Transfusion 14, 511 ( 1974). 14. Dayian, G., Reich, L., Mayer, K., Turc, J. M., and Rowe, A. W. Use of glycerol to preserve platelets suitable for transfusion. Cryobiology 11, 563 (1974). 15. Djerassi, I., Farber, S., Roy, A., and Cavins, J. Preparation and in u&o circulation of human platelets preserved with combined dimethyl sulfoxide and dextrose. Tmnsfusion 6, 572-576 ( 1966). 16. Handin, R. I., and Valeri, C. R. Improved viability of previously frozen platelets. Bbod 40, 509-513 ( 1972). 17. Jerushalmy, Z., and Zucker, M. B. Some effects of fibrinogen degradation products on blood platelets. Thomb. Diath. Haemorrhag. 15, 413 (1966). 18. Kim, B. K., and Baldini, M. G. Preservation of viable platelets by freezing. Effect of plastic containers. PYOC. SOC. Erp. Biol. Med. 142, 345 ( 1973). 19. Marcus, A. J., and Zucker, M. B. “The Physiology of Blood Platelets.” Grune and Stratton, New York, 1965. 20. McDonald, T. P., Odell, T. T., Jr., and Goslee, D. G. Platelet size distribution in relation to age. Proc. Sot. Exp. Biol. Med. 115, 684 (1964). 21. Murphy, S., Sayar, S. N., Abdou, N. L., and Gardner, F. H. Platelet preservation by freezing. Use of dimethyl sulfoxide as cryoprotective agent. Transfusion 14, 139144 ( 1974). 22. Rowe, A. W., and Peterson, J. Effect of glycerol, HES and DMSO on functional integrity of human blood platelets before and after freezing. Cryobiology 8, 393 ( 1971). 23. Valeri, C. R., Feingold, H., and Marchionni, L. D. A simple method for freezing human platelets using 6% dimethyl sulfoxide and storage at -80°C. Blood 43, 131-146 (1974).
