Platelet Preservation IV. Preparation and Cryopreservation of Platelet Concentrates from Fresh and Overnight-Stored Human Blood J. ODINK, J. J. F. M. DE WIT,
c.L. JANSSEN, AND H. K. PRINS
From the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service,
Amsterdam. The Netherlands
Two methods were used for the preparation of platelet concentrates from fresh and overnight-stored human ACD blood. In a two-step method prostoglandin E,was added to platelet rich plasma and the mixture was centrifuged to obtain platelet concentrate. The IBM 2991 Blood Cell Processor was used in a one-step-method. The concentrates were stored in the frozen state. Concentrates prepared with the two-step method had a higher platelet recovery and a lower leukocyte contamination than did concentrates prepared with the one-step method. Based on serotonin uptake velocity, response to hypotonic stress and available platelet lactor 3, no essential differences were observed between platelet concentrates prepared from fresh and overnight-stored blood. Concentrates prepared with the one-step method had a higher serotonin uptake velocity than those prepared with the two-step method. This did not result in a better recovery after cryopreservation.
I N O U R I N S T I T U T E large amounts of overnight-stored human blood are processed for the preparation of plasma products. Overnight-stored blood is ACD blood, collected in the evening by mobile teams, and stored overnight in a cold storage room (0 to 4C). At the time the processing starts the temperature of the blood is approximately 15 C. We were interested in whether platelets for transfusion purpose could also be prepared from this blood. A good procedure to prepare platelet concentrates is needed for several reasons: availability of plasma, small volumes to be transfused, and small volumes to be stored. This last aspect is especially impartant for cryopreservation since the smaller the volume the less cryoprotectant (DMSO) will be used and transfused. The classical method
to prepare platelet concentrates is centrifugation of whole blood to obtain platelet-rich plasma, followed by centrifugation of platelet-rich plasma (with or without addition of aggregation in hi bi tors): the two-step method. The IBM 299 1 Blood Cell Processor makes it possible to prepare platelet concentrates in one centrifugation step: the one-step method.? We thought it worthwhile to perform systematic studies on the use of fresh and overnight-stored blood for the preparation of platelet concentrates and on the quality of the concentrates before and after cryopreservation. We determined platelet recovery and leukocyte contamination, as well as the serotonin uptake and the response to hypotonic stress. T h e last two tests a r e considered to be viability criteria.9-17~20.21.23~z7 Available platelet factor 3 was determined since the infusion of large amounts of platelet factor 3 might be hazardous. In the two-step method prostaglandin El (PGE,) was added to platelet-rich plasma in order to prevent ir.reversible aggregation during the concentration step. Since P G E , might influence platelet recovery following cryopreservation, samples in the one-step method to which PGE was added before cryopreservation were also examined. Materials and Methods Phosphate-buffered salt solution contained 140 m M NaCI, 9 m M N a 2 H P 0 , and 1.3 m M NaH,PO, (pH 7.4). Prostaglandin E, (PGE,) was stored at 4 C as solution (100 pg/ml) in ethanol
Received for publication June 7, 1976; accepted January 22, 1977.
0041-1 132-78-0100-0021-0085 o J. B. Lippincott Co. Translusion Jan. Feb. 1978
Volume 18 Number I
Transfusion Jan. Feb. 1978
ODINK E T A L .
FIG. I . Effect of centrifugal force on the volume of the supernatant plasma (0-0, percentage of the volume of the original whole blood) and on the platelet yield in the supernatant (0-0, platelets x lo-’) after centrifugation for 25 minutes at room temperature of a bottle containing 500 rnl overnight-stored blood. The values are the mean of 1 1 experiments.
(99.8%), and diluted before use with phosphatebuffered salt solution. “C-serotonin ( 5 hydroxytryptamine-3’-C 14 creatinine sulphate; 57 Ci/mole) was obtained from The Radiochemical Centre, Amersham, UK, and diluted with ‘cold’ serotonin obtained from Merck, Darmstadt, GFR. Serotonin was stored at -4OC in phosphate-buffered salt solution. Russell’s viper venom (Stypven) was obtained freeze-dried from Wellcome Beckenham, UK, and stored at 4 C . Before use 0.1 mg was dissolved in I ml diluent. Substrate plasma for the determination of available platelet factor 3 was prepared as soon as possible after venipuncture by centrifugation of ACD blood for two minutes at 9,000 x g at room temperature. The plasma was stored in plastic tubes at room temperature. Scintillation fluid consisted of 450 ml toluene, 450 ml Triton-X 100, 9 ml acetic acid, 90 ml H,O and 9 g PPO (2.5diphenyloxazole). Dimethylsulfoxide (DMSO) was diluted with autologous plasma to 10% (v/v) and added rapidly at room temperature in a 1:l ratio to the platelet concentrates. Blood Collection and Storage Blood was collected in glass bottles, containing 80 ml ACD (acid citrate dextrose) solution (USP formula A). Overnight-stored blood was collected in the evening by mobile teams, and transported to the center in closed, wooden boxes, each containing 18 bottles of blood. The boxes were stored overnight (6 to 9 hours) in a cold storage room (0 to 4 C). Platelet Concentrates. Two units of either fresh or overnight-stored blood (two (ABO and rhesus identical donors) were pooled and subsequently divided into two portions. One portion was used for the one-step
method and the other for the two-step method. The one-step method was performed at room temperature by centrifugation of ACD blood at 3,000 rpm (1,000 x g ) in the IBM Blood Cell Processor. After 15 minutes pumping was started (during centrifugation), and a fraction of 10 ml was collected which contained a high concentration of platelets.’ The two-step method was performed as follows: 500 ml ACD blood was centrifuged in a glass bottle for 25 minutes at room temperature at 760 x g . This time and force was found to give the best yield of both platelets and plasma (Fig 1). Platelet-rich plasma was obtained by suction and centrifuged in a glass bottle for 20 minutes at 2,200 x g at room temperature after addition of PGE, to a final concentration of 10 ng/ml. The resulting pellet was resuspended in plasma (lj20 of the original volume). Cryopreservation One ml platelet concentrate was diluted rapidly at room temperature with 1 ml 10%(v/v) DMSO in plasma, and cooled from 0 to -100 C in 2 ml glass ampules as described p r e v i o ~ s l y .The ~~~~~ ampules were cooled for five minutes from 0 C with an external cooling rate of 1.6 degrees C/minute. The external temperature was then reduced to -35 C in order to induce the formation of ice and to remove the heat of fusion. After acheiving an internal temperature plateau cooling was continued, first at a cooling rate of 1.9 C/minute for seven minutes and then at 7.1 C/minute to - 100 C. The process was controlled by a Cryoson programmed biological freezer (BV4). The ampules were stored in liquid nitrogen (- 196 C). Thawing was performed by shaking the ampules in a waterbath at 40 C. After thawing the suspensions were diluted ten-fold with autologous plasma.
Volume 18 Number I
Platelet Studies Serotonin uptake velocity was expressed as the initial rate of disappearance of serotonin from the supernatant fluid of a platelet suspension. For this purpose platelet suspensions (7 ml) were incubated at 3 7 C . After five minutes 70 pl serotonin solution (568 pM, 1.86 dpm/pmole) was added. Every 45 seconds thereafter (during 7.5 minutes) a sample (0.6 ml) was taken from which the platelets were spun down in an Eppendorf centrifuge (model 3200) for 1.5 minutes at 9,000 x g at room temperature. A 0.5 ml sample of the supernatant fluid was mixed with 10 to 12 ml scintillation fluid. Counting was performed with a liquid scintillation counter (85% efficiency)., The determination of the serotonin uptake velocity of cryopreserved platelet suspensions was performed in duplicate. The specific activity (dpm/mole) of the serotonin present in the sample during the performance of the assay was not exactly known, since we did not determine the amount of serotonin present in the plasma before the addition of '%-serotonin (- 1/20 of the amount which was added). Therefore, the serotonin uptake velocity was expressed as dpm/108 cells/minute. Response to hypotonic stress was determined by recording the absorbance at 610 nm after mixing 1.5 ml platelet suspension with 0.5 ml H,O in a 3 ml cuvette with 1 cm lightpath at room temperature against a blank of 1.5 ml platelet suspension with 0.5 ml NaCl (154 mM). The result was expressed as V,,, /A m,n (min-').zo The determination of the response to hypotonic stress of cryopreserved platelet suspensions was performed in duplicate. Serotonin uptake velocity and response to hypotonic stress of the platelet concentrates were always determined after 20-fold dilution with autologous plasma. Platelet concentrates containing 5 % (v/v) DMSO were diluted 10-fold before these parameters were determined. Available platelet factor 3 was determined from a 0.1 ml sample (diluted with autologous plasma to 200 platelets/nl) added to 0.1 ml substrate plasma in a glass tube and incubated for one minute at 37 C. Then 0.1 ml Russell's viper venom (0.1 mg/ml) and 0.1 ml CaCI, (25 mM) were added and the clotting time was determined. The avail-
able platelet factor 3 was expressed as the ratio [(log a/b)/(log a/c)] x 100, where a = clotting time of the substrate, b = clotting time of the sample, and c = clotting time of the sonicated sample (i.e., 0 is the value of the substrate plasma and 100 is the value of the sonicated sample). Sonication was performed for 30 seconds (lo5 cycles/sec). Prolongation of the sonication time did not shorten the clotting time. Cell counting was performed with the Codter counter (model ZF; 50 pm orifice for platelets, 100 pm orifice for leukocytes). Statistics
P values for differences in serotonin uptake v e locity, response to hypotonic stress, and available platelet factor 3 between fresh and overnightstored blood were calculated with Student's t-test for the difference of two sample means. P values for other differences in the values of these parameters were calculated with Student's t-test for paired observations, since these values were obtained from the same series of experiments. P values for differences in platelet recovery and leukocyte contamination between fresh and overnight-stored blood were calculated with Wilcoxon's signed rank test for paired observations. Differences were considered to be significant if PD < 0.05.
Results The overnight-stored blood was maintained at an ambiant temperature of 0 to 4 C for six to nine hours but the blood had not cooled to this temperature at the time the experiment started. The mean temperature of the overnight stored blood was 15 C (range: 13 to 18 C). The average platelet yield in the concentrates prepared with either method is shown in Table 1. It appeared that platelets could be collected from overnight-stored blood in at least the same amount as from fresh blood. The platelet recovery in the concentrates prepared from fresh blood was lower (PD < 0.001) with the one-step method than with the two-step method. This might be due to microscopic aggregates which were present in
Table 1. Effect of the Preparation Method on ?heNumber of Pfatefets Expressed as 1 0 s Platelets in Platelet Concentrates From 500 ML of Blood
Fresh Blood (n 12) Isolation Procedure Two-step method One-step method 'Standard deviation.
Overnight-Stored Blood (n 14)
58-1 1 1 35-97
47-1 71 59-122
Transfusion Feh. 1978
ODINK E T A L Table 2.
Effect of the Preparation Method on the Number of Leukocytes (Expressed as loe Leukocytes) in Platelet Concentrates from 500 MI of Blood
Fresh Blood (n = 11) Isolation Procedure Two-step method One-step method
Overnight-Stored Blood (n = 12)
66 51 6
9-196 84-2.1 85
several concentrates prepared with the one-step method from fresh blood. In some experiments almost complete irreversible aggregation occurred. When this happened the experiment was stopped and rejected. The average leukocyte contamination was higher in the concentrates prepared with the onestep method than in the concentrates prepared with the two-step method (Table 2). However, this difference was only significant (PD < 0.001) when overnight-stored blood was used. The influence of the different steps in the preparation and cryopreservation of platelet concentrates on serotonin uptake velocity, response to hypotonic stress and available platelet factor 3 is shown in Tables 3,4, and 5 respectively. The first two parameters were determined in the same series of experiments. The method of preparation of concentrates from platelet-rich plasma hardly influenced response to hypotonic stress and available platelet factor 3. The observed decrease in serotonin uptake velocity was only significant when fresh blood was used (PD < 0.001). This is most likely due to the fact that the platelet concentrate had been at room temperature for one to two hours before serotonin uptake Table 3.
velocity was determined.23 It was not clear how the addition of PGE, caused an increase in response to hypotonic stress compared with plat elet-rich plasma when overnigh t-s tor ed blood was used (PD < 0.05). Minimal differences were observed in response to hypotonic stress and available platelet factor 3 between the concentrates with respect to preparation method and the history of the blood used. Serotonin uptake velocity appeared to be higher when concentrates were prepared with the one-step method. However, this difference appeared to be only significant when overnight-stored blood was used (Po < 0.05). Cryopreservation of platelet concentrates resulted in a considerable loss in serotonin uptake velocity and response to hypotonic stress. It resulted in a considerable increase in available platelet factor 3, while no decrease in platelet number was observed. The presence of PGE, during cryopreservation did not improve the recovery after cryopreservation. There appeared to be no difference after cryopreservation in serotonin uptake velocity, response to hypotonic stress and available platelet factor 3 of platelet concentrates prepared from
Effect of the Different Steps in the Preparation and Cryopreservation of Platelet Concentrates on Serotonin Uptake Velocity ldpm/ 10%cells/minl
Overnight-Stored Blood (n = 12)
Fresh Blood (n = 12)
Two-step method Platelet-rich plasma Platelet-rich plasma Platelet concentrate Platelet concentrate Cryopreservation
+ PGE , + DMSO
One-step method Platelet concentrate Platelet concentrate DMSO Plateletconcentrate DMSO Cryopreservation Cryopreservation PGE,
169 172 148 125 48
29 34 29 31 11
1 19-220 1 19-232 109-204 86-1 99 30-73
155 159 148 139 41
34 29 33 22 9
83-2 10 1 1 7-225 102-228 94-1 78 32-62
164 140 132 52 47
45 29 29 12 16
97-257 85-1 94 88-1 79 43-86 35-88
180 153 152 36 36
47 18 27 10 7
123-305 1 24-1 8 1 1 17-204 17-59 24-50
Volume 18 Number I
Effect of the Different Steps in the Preparation and Cryopreservation of Platelet Concentrates on Response to Hypotonic Stress lL&ax/Amin;min-') See Ref. 20 Fresh Blood (n = 12)
Two-step method Platelet-rich plasma Platelet-rich plasma Platelet concentrate Ptatelet concentrate Cryopreservation
One-step method Platelet concentrate DMSO Platelet concentrate Platelet concentrate DMSO Cryopreservation Cryopreservation PGE,
Overnight-Stored Blood (n = 12)
0.43 0.45 0.40 0.32 0.17
0.06 0.06 0.10 0.07 0.06
0.35-0.55 0.38-0.57 0.25-0.62 0.23-0.46 0.10-0.28
0.39 0.47 0.41 0.41 0.15
0.09 0.16 0.09 0.09 0.05
0.264.58 0.34-0.91 0.26-0.56 0.23-0.58 0.10-0.24
0.42 0.37 0.33 0.15 0.15
0.10 0.09 0.07 0.05 0.06
0.29-0.58 0.22-0.54 0.24-0.44 0.08-0.25 0.08-0.27
0.41 0.34 0.38 0.09 0.1 1
0.08 0.08 0.09 0.04 0.04
0.25-0.53 0.22-0.45 0.19-0.55 0.01-0.15 0.05-0.19
* Standard deviation.
fresh blood with respect to the preparation method. However, concentrates prepared from overnight-stored blood with the one-step method when compared concentrates prepared from overnight-stored blood with the two-step method showed a lower recovery of serotonin uptake velocity (PD < 0.05) and response to hypotonic stress (P., < 0.001), and a higher level of available platelet factor 3 (Po < 0.05) after cryopreservation (without the presenceof PGE,).
Discussion In the early days of platelet transfusion smooth concentrates were prepared by using EDTA as anticoagulant. However, it soon Table 5.
appeared that this agent drastically reduced platelet recovery in vivo as compared with ACD.I.3.6.18 T h e use of EDTA can be avoided, either by using extra ACD as anticoagulant3 or by the addition of ACD to platelet-rich plasma before the concentration ~ t e p . ~ * ~Both . ' ~ methods * ~ ~ . ~have ~ the disadvantage of decreasing the yield of factor VIII due to the lower pH of the plasma caused by the use of extra ACD.24 M ~ n r a d 'showed ~ that the use of extra ACD could be avoided by centrifuging platelet-rich plasma at 25 C and incubating the centrifuged platelets before resuspension for 30 minutes at this
Effect of the Different Steps in the Preparation and Cryopreservation of Platelet Concentrates on Available Platelet Factor 3 (W Overnight-Stored Blood (n = 71
Fresh Blood (n = 4)
34 38 39 42 90
15 10 13 12 2
19-54 33-53 32-59 31-59 88-92
49 46 46 54 84
12 12 12 11 8
24-60 20-57 23-58 33-66 73-98
41 44 47 93 89
14 9 6 4 5
2 1-52 33-53 40-54 88-97 84-96
51 48 53 92 94
13 12 12 7 7
28-69 28-67 30-65 83- 100 78-98
Mean Two-step method Platelet-rich plasma Platelet-rich plasma Platelet concentrate Platelet concentrate Cryopreservation
One-step method Platelet concentrate DMSO Platelet concentrate Platelet concentrate DMSO Cryopreservation Cryopreservation PGE,
ODlNK E T A L .
temperature. Apart from the fact that this method is more time consuming, it has the disadvantage that a successful resuspension of the platelet pellet is not always ensured. The use of PGE, in blood banking for the preparation of platelet concentrates was proposed by Shio and RamwelLZ5The addition of PGE, to whole blood after collection improved the recovery of platelets from fresh blood and from blood stored for several day^.^*^*^^ Platelet survival in vivo and hemostatic effectiveness of platelet suspensions are not influenced by the treatment with PGE,.4*28 Using 10 ng PGE,/ml we have not observed any problems in resuspending platelets after centrifugation. Besides the favorable aspects of the use of PGE,, the agent has the disadvantage of being unstable in aqueous solutions, especially when the pH of the solution is high (pH > 8) or low (pH < 7). This implies that it cannot be preserved in the anticoagulant (ACD solution) without loss in activity. Furthermore, there might be a risk of infusing PGE, into a patient. However, when platelet concentrates are prepared for transfusion purposes the volume of one unit platelet-rich plasma can easily be reduced to 10 ml. This results in the administration of 1 pg PGE, for each concentrate which will be transfused. No toxic side effects of such amounts of PGE, have been reported. It is not known whether plasma fractionation results in a accumulation of PGE, or its degradation products in a specific fraction. At present acidification of platelet-rich plasma before centrifugation and the method of MonradI9 are the most commonly used procedures for the preparation of platelet concentrates for transfusion purpose. I n spite of this and of the fact that PGE, cannot yet be used in routine blood banking, we decided to use this agent in this study since it is highly reliable for the rapid preparation of p!atelet concentrates. The use of PGE, for the preparation of platelet concentrates with the two-step method from overnight-stored blood is in most cases not required, since
Transfusion Jan. Feb. 1978
these platelets are less sensitive to aggregation inducing stimuli (authors’ unpublished observations). We did use it, however, in order to avoid any resuspension problems, and to allow comparison between platelet suspensions prepared from fresh and overnight-stored blood. The one-step method had the advantage of a rapid preparation of a single concentrate, but when more concentrates are wanted the two-step method is preferable since the IBM 2991 Blood Cell Processor can be used only for one unit at a time. The concentrates prepared with this method showed a higher serotonin uptake velocity (Table 3) when compared to the concentrates with the twostep method. However, the platelet recovery was lower and the leukocyte contamination was higher. Furthermore, t h e one-step method has the disadvantage that there is a risk that complete irreversible aggregation may occur when fresh blood is used. The occurrence of irreversible aggregates in the concentrates prepared with the one-step method is probably caused by the rather narrow outlet from the circular bag into the tubing system, and by the fact that the temperature of the rotating seal rises during centrifugatkn (the centrifuge cannot be cooled.)’ Irreversible aggregation did not occur when overnight-stored blood was used. These platelets appear to be less sensitive to aggregation inducing stimuli. The high leukocyte contamination in the concentrates prepared from overnight-stored blood with the one-step method was not unexpected. A considerable overlap of the distribution curves of the platelets and the leukocytes was found by de Wit et af.’ at the interface of plasma and erythrocytes after centrifugation. The occurrence of aggregates when fresh blood was used may have reduced the contamination by including the leukocytes in the aggregates. T h e motive to determine t h e initial serotonin uptake velocity a t one fixed concentration and the consequences of it have been discussed elsewhere.23 Wez3 ob-
served, like Hardeman and Heynens,I2a fair correlation between changes in this parameter and the response to hypotonic stress. The most important changes in the serotonin uptake velocity, the response to hypotonic stress, and the available platelet factor 3 were caused by cryopreservation. These effects of cryopreservation are consistent with the findings in other studies from this l a b ~ r a t o r y . From ~ ~ * ~the ~ data shown in Tables 3, 4, and 5 it may also be concluded that the slight differences before cryopreservation did not influence the recovery after cryopreservation. Furthermore, the obtained results were hardly influenced by the presence of PGE,. The importance of the observed changes after cryopreservation with respect to platelet survival in vivo and the possible occurrence of hypercoagulability after infusion now require clincial investigation. Preservation at 4 C, even for a few hours,2 decreases the serotonin uptake velocity and the response to hypotonic stress of platelets.g*12*i8 Therefore, we did not expect that the platelet concentrates prepared from overnight-stored blood would be at as good as those prepared from fresh blood. The temperature of overnight-stored blood did not reach a temperature at which damage occurs, and/or that the slow fall in temperature enabled the platelets to adapt to the changes in their environment. However, when cryopreservation was also considered, platelet concentrates prepared from overnight-stored blood appeared to be less favorable, although the observed differences were not significant. In conclusion it may be stated that for the preparation of platelet concentrates the two-step method is preferable, while it is worthwhile to evaluate the clinical effectiveness of plat el et concentrates prepared from overnight-stored blood. Acknowledgments The authors wish to thank Miss Willeke van Leeuwen, Messrs. H. P. J. Henrichs and R. Sprokholt for their skillful technical assistance. The prostaglandin El was a
gift from Unilever Research, Vlaardingen, the Netherlands.
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Lundberg, A., and S. Murphy: Survival in vivo of human blood platelets frozen without protective additives. Scand. J. Haematol. 9:222, 1972. 18. Morrison, F. S., and M. Baldini: The favorable effect of ACD on the viability of fresh and stored human platelets. Vox Sang. 12:90, 1967. 19. Monrad, N.: A simple method for obtaining platelet concentrates free of aggregates. Transfusion 8:48, 1968. 20. Odink, J.: Platelet preservation. 11. The response of human platelet suspensions to hypotonic stress. Thrombos. Diathes, Haemostas. 36:182,
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J. Odink, Ph.D., Research Associate, University Hospital, Department of Obstetrics and Gynaecology, Rijnsburgerweg 10, Leiden. J. J. F. M. d e Wit, Technician, Free University, Pediatric Laboratory, De Boeleaan I 117, Amsterdam. Catherine L. Janssen, Ph.D.. AVSH (Training of chemical technicians), Bennebroekstraat 1 1- 13, Amsterdam. H. K. Prins, Ph. D., Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, Amsterdam.