Vox Sang. 30: 401411 (1976)

Storage of Human Platelets by Freezing1 B. K. KIM, K. TANOUE, and M. G. BALDIN1 Division of Hematologic Research, The Memorial Hospital, Pawtucket, and Brown University, Providence, R. I.

Ahstruct. Prolonged, probably indefinite storage of viable and functional human platelets is now possible by freezing with dimethylsulfoxide (DMSO). These platelets have a nearly normal survival upon reinfusion and are capable of sustained hemostatic effectiveness in thrombocytopenic patients. Adaptation of the freezing technique for large-scale usage has niore recently been achieved. The method is mainly based on the following principles: (1) use of plasma for suspension of the platelet concentrate; (2) gradual addition ( 0 5 % every 2 min) of DMSO to a final concentration of 5 % and its gradual removal; (3) a slow cooling rate of about 1 “C per min and rapid thawing (in ‘I min); (4) use of a polyolefin plastic bag for freezing; ( 5 ) a washing medium of 20% plasma in Hanks’ balanced salt solution; ( 6 ) final resuspension of the platelets in 50% plasma in Hanks’ solution.

The long search for an efficient method of platelet preservation by freezing has recently been attended with success. We believe that in the near future this event will be of significant consequence in the treatment of acute and chronic thrombocytopenic patients because of the following considerations: ( 3 ) As recently demonstrated, frozen human platelets can arrest hemorrhage in thrombocytopenic patients [9, 10, 191. This has made the long-term preservation of human platelets available for clinical use while it is now well recognized that by storage at non-freezing temperatures (4 ‘’ or 22 C ) human platelets can be preserved viable (i.e., capable to recirculate and survive upon infusion) and functional (i.e., with normal 1 This work was supported by the US Atomic Energy Commission, contract AT(l1-1)-3234.

Received: June 20, 1975; accepted: July 16, 1975.

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cell functions and in vivo effect on hemostasis) for only a few hours. It is known that after 1 day at 4 OC platelets have lost viability t o a large extent [17]; after the same period of time at room temperature (22 "C) the platelets have lost aggregability and, in part, hemostatic effectiveness [3,6, 181. (2) With the advent of blood component therapy, the availability of an efficient method for the long-term preservation of human platelets will make available to clinical practice very large quantities of platelets which are presently not utilized. Platelets could be harvested and stored as a byproduct of blood banking. (3) It is now known that platelet isosensitization, a phenomenon which can drastically reduce the effectiveness of platelet transfusion therapy in patients [l], can be prevented to a large extent by the use of HL-A type-specific platelets [21]. This will in the future expand the use of platelet transfusions to chronic thrombocytopenic patients. (4) Autologous platelet transfusions can now also be utilized. This is done by freezing the patient's own platelets collected prior to the occurrence of thrombocytopenia and is particularly useful in selected patients, e.g. in patients scheduled to receive chemotherapy for a malignant process. The availability of a cell separator by which large volumes of platelets can be collected from one single donor in a relatively short period of time, will ease the procedure. (5) The use of frozen platelets may lessen the risk of serum hepatitis from platelet transfusions since this is apparently true with frozen red cells 181. (6) A national or international pool of frozen platelets could be organized and probably stored indefinitely for use in special emergencies, as in the case of an atomic disaster. Early attempts at freczing human platelets were largely unsuccessful because by freezing and thawing platelets lost the capacity to recirculate and survive upon infusion [2, 4, 5, 14, 15, 17, 201, an essential prerequisite for the platelets to be effective in hemostasis. We and others [7, 11, 121 have recently demonstrated that (1) when osmotic stress is minimized, human platelets can be frozen and thawed by the use of 5 % dimethylsulfoxide (DMSO) in plasma with practically no damage to their viability, i.c., their capacity to recirculate and survive upon infusion. 'This is done by regulating the addition and the removal of DMSO to the platelet suspension so that the change in concentration is 0.5% (or less) every 2 min; (2) the frozen platelets are effective in hemostasis and can

Storage of Human Platelets by Freezing

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Fig. I. Platelet response to hypotonic stress measured by the changes in light absorbancy at 420 nm. The rise in light absorbancy occurring during the first 2 min of the recovery phase was the value used for comparison and was expressed in percent of the control value.

shorten the prolonged bleeding time in thrombocytopenic patients 191; (3) the method has recently been rendered practical and suitable for the large-scale application in clinical practice. During the past few years we have experienced that the platelet contractive response to the swelling which occurs after exposure to hypotonic medium is a good indicator of platelet viability after storage in various conditions [11, 131. In our experiments, the hypotonic shock was produced by the addition of an equivolume of distilled water to the platelet suspension in plasma. Changes in the platelets were measured by the changes in light absorbancy in a recording spectrophotometer at 420 nm wavelength. As demonstrated by FANTI.in 3968, after the addition of water there is first a sudden decrease in light absorbancy which is followed by a gradual rise towards the original level. This is interpreted as due to a contraction of platelet volume after an original expansion following exposure to the hypotonic medium and is called, for convenience, 'reversal reaction' (fig. 1). We have shown that the platelet reversal reaction becomes progressively suppressed by storage of the platelets at 4 ' C or at 22 "C and follows closely the results obtained by the combersome method of in vivo platelet survival after labeling with Cr5*[13]. Using this practical in vitro technique, we first demonstrated [ I11 that DMSO was the cryoprotective agent of choice for the freezing of human plate-

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Fig. 2. Survival curves of human platelets preserved for short intervals by freezing with 5% DMSO. To simplify graphic representation, the first recovery values obtained 5 min post-infusion were not connected with the rest of the respective curves. Shadowed area represents the range of survival curves obtained with normal, fresh human platelets.

lets, that addition and removal of DMSO had to be done very gradually to minimize the osmotic stress to the platelets, and that the plastic container used for freezing was of importance since the usual polyvinylchloride bags appeared to reduce platelet viability while polyolefin bags did not. These results were later confirmed by viability studies of frozen platelets performed in human volunteers by “Cr-labeling (fig. 2) [12]. In our experiments, the radioactive label was added to the platelet concentrates before freezing and autologous survival was measured. The peak recovery value of platelet radioactivity in the circulation was on the average 63% (the normal value with freshly prepared platelets is 66%). The survival time of the previously frozen platelets was normal or nearly so in all experiments. Other studies confirmed the importance of the type of plastic used in the manufacture of containers for platelet freezing and storage [lZ]. Platelets frozen in Unitag bags made of polyvinyl-chloride (Abbott Laboratories) had a significantly reduced in vivo viability in comparison to similar platelet preparations frozen in Heinoflex bags made of polyolefin (Union Carbide Corp.). The reasons for this difference between the

Storage of Human Platelets by Freezing

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Fig. 3. Studies of platelet function before and after freezing are expressed in percent of the mean control value obtained with fresh platelets suspended in plasma.

two plastic containers is not presently known. The fact that polyolefin containers are manufactured without the use of plasticizers which like di-2-ethylhexyl phthalate are known to be present in polyvinylchloride bags and may leach off the plastic during freezing, thawing or storage, may be the main reason, but this has not yet been demonstrated experimentally. Although survival of previously frozen platelets is almost completely preserved by our method of freezing with 5% DMSO, platelet integrity is not totally saved by this technique. After preservation of human platelet concentrates at -79 "C for 1-4 days, we recently measured a few parameters of platelet biochemistry and function [9]. These included determination of the glycogen content, in vitro serotonin uptake, platelet aggregation with ADP and collagen, and quantification of platelet p-glucuronidase (a lysosomal enzyme) and purine nucleoside phosphorylase (PNP; a cytoplasmic enzyme). Freezing and thawing as described in our method, caused an average loss of 13% of platelet aggregation with ADP and of 36% of collagen-induced aggregation (fig. 3). Simple exposure of the platelets to DMSO without freezing and thawing caused no significant loss of platelet aggregation. The in vitro serotonin uptake was 38% of the control value, but it was not different from the control value before freezing and thawing, indicating that the DMSO

406 Glycogen content

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Fig. 4. Biochemical studies of platelets before and after freezing. Values are expressed in percent of the mean control value obtained with fresh platelets suspended in plasma.

per se had no detectable effect on this parameter. Similar moderate reductions in values were seen for the platelet glycogen content, p-glucuronidase and PNP-ase (fig. 4). The enzyme activities lost by the platelets were recovered in the suspending medium. In spite of these biochemical and functional alterations, the frozen platelets still had significant hemostatic effectiveness. This was demonstrated by the shortening of the bleeding time obtained in eleven thrombocytopenic patients in whom the frozen platelets were infused (table I) [9, 101. These were patients with severe and stable thrombocytopenia due to bone marrow failure either secondary to chemotherapy for leuke-

Tnb/e I. Bleeding time values before and after infusion of frozen platelets in thrombocytopenic patients Normal

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Storage of Human Platelets by Freezing

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Fig. 5. Increments in platelet count after infusion of frozen platelets. The control values (left) refer to increments obtained with fresh platelet concentrates.

mia or to idiopathic hypoplastic anemia. The platelet counts in the patients varied from 16,000 to 40,000 per mm3 before infusion of the frozen platelets. Nine of the patients studied had a bleeding time (done by the template method [16] longer than 25-30 min and the other two had a bleeding time of 39 and 21 min, respectively (normal 4-8 min). The infusion of the frozen platelets caused an immediate and significant increase in the platelet count in all the patients. At 15 min after infusion the mean platelet increment (related to the infusion of 1011 platelets per m2 of body surface (fig. 5) was 14,700 platelets per mm3 and was 12,000 per mm3 after 3 h and 5,500 per mm3 24 h post-infusion. The values were similar to those obtained with the infusion of freshly prepared platelet concentrates (fig. 5 ) and indirectly confirmed that the frozen platelets had normal viability in the circulation. Surprising was the fact that the bleeding time measured at 15 min post-infusion (table 1) was remarkably shorter than before infusion, but the value became lower 3 h post-infusion, reaching an average value of 9.5 min which was almost within the normal range. The bleeding time was still shorter after 24 h than before infusion, with a mean value of 19 min in eight patients, while it was longer than 25 rnin in one and more than 30 min in two other patients. These experiments demonstrated that immediately after infusion of the frozen platelets the maximal numerical increment did not correspond to the maximal effect on hemostasis which was recorded only 3 h post-infusion. This confirmed that the frozen platelets have a functional lesion,

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Table II. Platelet number and viability after washing the thawed platelet concentrates with different media Washing medium

Plasma 17% plasma +saline 1 1 Yo plasma + HBSS 20% plasma + HBSS

Recovery Yo

T1/,

V.1.'

days

%

Platelet number %

49.8 42.1 46.6 47.3

3.1 2.9 3.0 3.2

13.4 56.7 64.3 70.5

87.9 76.4 78.1 79.2

V.I. =Viability index derived by measuring the surface area under the platelet survival curve and expressed in percent of the value obtained with fresh platelets.

but it also proved that in the circulation the platelets can rapidly recover from this lesion reaching a higher hemostatic effectiveness within 3 h. We have concluded, therefore, that human platelets subjected to freezing with 5% DMSO have a nearly normal survival and are capable of sustained hemostatic effectiveness. These results are of practical significance and indicate that the long-term storage of viable human platelets is now possible. Our more recent studies have concentrated on a few technical problems in the attempt of rendering the method suitable for the large-scale application in clinical practice. When autologous platelet-poor plasma is used as the washing medium for the removal of DMSO after thawing of the platelet concentrate, simultaneous storage of a large volume of autologous plasma becomes necessary. We have recently examined whether plasma is indispensable for washing the previously frozen platelets. In a series of experiments, the frozen platelets were thawed, then washed once with 11% plasma in Hanks' balanced salt solution (HBSS) containing phosphate, bicarbonate, potassium and glucose; in other experiments, they were washed with 17% plasma in saline. The platelets washed in plasma-saline had significantly lower viability values in vivo as demonstrated by a lower recovery (42%) in the platelet survival curve (50% in the control), and a reduced platelet number in the concentrate after washing (table 11). With 11% plasma in HBSS the values were somewhat improved. With 20% plasma in HBSS, results were similar to those obtained with a pure-plasma washing. We have concluded, therefore, that in the washing medium this amount of plasma is

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Fig. 6. A new polyolefin plastic bag for freezing and storage of human platelets i s now available (Union Carbide Corporation, No. 2031-4) in which a small compartment for the storage of 20 ml plasma is separated from the major compartment in which the platelet concentrate is stored.

indispensable. Since the platelet concentrate is suspended in 30 ml plasma, the addition of 120 ml HBSS is advised for removal of the DMSO. One additional finding that we obtained in these experiments is that the washed platelet concentrate must finally be resuspended in 50% plasma in HBSS for reinfusion. For this purpose an amount of 20 ml of autologous plasma must always be stored with the platelet concentrate. Our recently designed polyolefin bag produced by the Union Carbide Corporation (No. 2031-4) (fig. 6) provides a double compartment, one for storage of the platelet concentrate and one for storage of the 20 ml plasma. This container is practical and provides the necessary, safe portals for the maintenance of sterility in the bag. These details have now made the method available for the wide application in clinical practice in those countries in which no ban against

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the injection of traces of DMSO exists. In the US, on the other hand, this ban still persists and presently inhibits the wide application of the method until we will prove by controlled clinical experiments that the traces of DMSO present in the final platelet concentrates are absolutely safe for the patients receiving single or multiple platelet transfusions. The present ban is based on the fact that large and prolonged doses of DMSO are known to cause significant side effects in animals. However, no side effects have so far been seen in humans and we predict that in the near future the method will be in common clinical use in the US.

References I BALDINI, M.; COSTEA,N., and EBBE,S.: Studies on the antigenic structure of blood platelets. Proc. 8th Congr. Eur. SOC.Hematol., pp. 378-383 (Karger, Basel 1962). 2 BALDINI, M.; COSTEA, N., and DAMASH~K, W.: The viability of stored human platelets. Blood 16: 1669 (1960). 3 BECKER, R. I.; TUCCELLI, M.; KUNICKI,T.; CHALOS,M. K., and ASTER,R. H.: Studies of platelet concentrates stored at 22°C and 4°C. Transfusion, Philad. 13: 61 (1973). F. H.: Thrombocytopenic bleeding and preservation of 4 COHEN,P. and GARDNER. platelets; in Blood platelets, p. 485 (Little, Brown, Boston 1961). 5 DJERASSI, I.; FARBER, S.; ROY,A., and CAVINS, J.: Preservation and in vivo circulation of human platelets preserved with combined dimethylsulfoxide and dextrose. Transfusion, Philad. 6: 572 (1966). R. I. and VALERI, C. R.: Hemostatic effectiveness of platelets stored at 22°C. 6 HANDIN, New Engl. J. Med. 285: 538 (1971). C. R.: Improved viability of previously frozen platelets. 7 HANDIN,R. 1. and VALERI, Blood 40: 509 (1972). 8 HUGGINS, C. E.: Frozen blood - clinical experience. Surgery, St Louis 60: 77 (1966). M. G.: Biochemistry, function and hemostatic effectiveness 9 KIM,B. K. and BALDINI, of frozen human platelets. Proc. SOC.exp. Biol. Med. 145: 830 (1974). 10 KIM,B. K. and BALDINI, M. G.: Storage of human platelets by freezing; in BALDINI and EBBEPlatelets: production, function, transfusion and storage (Grune & Stratton, New York 1974). 1 1 KIM,B. K . and BALDINI,M. G.: Reversal reaction and viability of frozen human platelets. Abstract. Cryobiology 9: 329 (1972). 12 KIM,B. K. and BALDINI, M. G.: Preservation of viable platelets by freezing. Effect of plastic containers. Proc. SOC.exp. Biol. Med. 142: 345 (1973). 13 KIM,B. K. and BALDINI, M. G.: The platelet response to hypotonic shock. Its value as an indicator of platelet viability after storage. Transfusion, Philad. 14: 130 (1974). S.; FREEMAN, G., and FIORENTINO, R.: Hemostasis in 14 KLEIN,E.; TOCH,R.; FARBER, thrombocytopenic bleeding following infusion of stored, frozen platelets. Blood 11: 693 (1956).

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15 LUNDBERG, A.; YANKEE, R. A.; HENDERSON, E. S., and PERT,J. H.: Clinical effectiveness of blood platelets preserved by freezing. Transfusion, Philad. 7: 380 (1967). 16 MIELKE, C. H., jr.; KANFSHIRO, M. M . ; MAHER, I. A.; WEINER, J. M., and RAPAPORT, S. 1.: The standardized normal Ivy bleeding time and its prolongation by aspirin. Blood 34: 204 (1969). F. and BALDINI, M. G . : The favoiable effect of ACD on the viability of 17 MORRISON, fresh and stored human platelets. Vox Sang. 12: 90 (1967). 18 MURPHY, S. and GARDNER, F. H.: Platelet storage at 22°C; metabolic, morphologic. and functional studies. J. din. Invest. 50: 370 (1971). C. R. and FEINGOLD, H.: Hemostatic effectiveness of liquid preserved platelets 19 VALERI, stored at 4°C or 22°C and freeze-preserved platelets stored with 5% DMSO at -150°C or stored with 6% DMSO a t -80°C; in BALDINI and ERBEPlatelets: production, function, transfusion and storage (Grune & Stratton, New York 1974). 20 WEISS,A. J. and B A L L I N GW. ~ K F., , jr.: Feasability of storage of intact platelets with apparent preservation of function. Ann. Surg. 148: 360 (1958). 21 YANKEE,R. A.; GRAFF,K. s.; DOWLING, R., and HENDtRSON, E. s.: Selection Of unrelated compatible platelet donors by lymphocyte HL-A matching. New Engl. J. Med. 288: 760 (1973).

Dr. M. G. BALDINI,MD, Division of Hematologic Research, The Memorial Hospital, Pawtuckel, R.I. (USA)

Storage of human platelets by freezing.

Prolonged, probably indefinite storage of viable and functional human platelets is now possible by freezing with dimethylsulfoxide (DMSO). These plate...
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