New plasma-reduced synthetic media, Fukushima cocktails, for the storage of platelets for transfusion

Transfusion Science 23 (2000) 37±46

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New plasma-reduced synthetic media, Fukushima cocktails, for the storage of platelets for transfusion Takeshi Yuasa a, Hitoshi Ohto a,*, Akira Suzuki b, Fumio Shishido b a

Division of Blood Transfusion and Transplantation Immunology, School of Medicine, Fukushima Medical University, Hikariga-oka Fukushima City, Fukushima 960-1295, Japan b Department of Radiology, School of Medicine, Fukushima Medical University, Hikariga-oka Fukushima City, Fukushima 960-1295, Japan Received 9 December 1999; accepted 23 February 2000

Abstract Background. Donor plasma proteins are associated with non-hemolytic allergic reactions, such as urticaria or laryngeal edema, in platelet-transfusion recipients. Replacement of plasma with synthetic media from platelet concentrates (PCs) is considered to be e€ective in preventing such reactions. However, platelets preserved in media depleted of less than 10% plasma are reported to have functions inferior to those preserved in plasma. Methods. Fukushima Cocktails (FCs) contain glycerol (25, 50 or 100 mM), sodium acetate, glucose and other components. To test the e€ect and determine the most suitable concentration of glycerol for platelet preservation, functions of platelets including aggregation, hypotonic shock response and swirling pattern and released biochemicals were measured with platelets preserved in Fukushima Cocktails. The e€ects of residual plasma on platelet functions were also evaluated. Autologous platelets stored for 3 days in solution containing 50 mM glycerol were transfused into healthy volunteer donors to evaluate their safety and survival. Results. The functions (aggregation and hypotonic shock response) of platelets preserved in Fukushima Cocktails with 10% residual plasma were preserved for 5±7 days as well as plasma controls, whereas platelets stored for 9 days in a medium lacking glycerol became swollen and b-thromboglobulin and thromboxane B2 increased. When the residual plasma was more than 5%, platelet functions including aggregation, hypotonic shock response and swirling pattern were well preserved for 7 days. The in vivo platelet survival rates at 24 and 48 h after transfusion of platelets stored for 3 days in Fukushima Cocktail were 77% and 60%, respectively, which were not less than autologous plasma-stored platelets. Conclusion. Glycerol at a concentration of around 50 mM has a bene®cial e€ect on platelet preservation for more than 7 days. The results of these experiments indicate that platelets stored in Fukushima Cocktail should be useful clinically. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Platelet; Preservation medium; Platelet function; Platelet survival

*

Corresponding author. Tel.: +81-245-48-2111; fax: +81-245-49-3126. Abbreviations: HSR ˆ hypotonic shock response; PC(s) ˆ platelet concentrate(s); FC ˆ Fukushima Cocktail; PTR ˆ platelet transfusion refractoriness; MPV ˆ mean platelet volume; b-TG ˆ b-thromboglobulin; TXB2 ˆ thromboxane B2 . E-mail address: [email protected] (H. Ohto). 0955-3886/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 5 - 3 8 8 6 ( 0 0 ) 0 0 0 4 6 - 1

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T. Yuasa et al. / Transfusion Science 23 (2000) 37±46

1. Introduction The performance of platelet concentrates (PCs) in transfusion is greatly limited by a short (5 days internationally and 3 days in Japan) shelf life. Many blood banks frequently face a shortage of PCs, when supply cannot keep up with demand and, at other periods, large numbers of PCs are wasted because they cannot be used before their expiration date. During preparation and storage, platelets manifest both as a loss of function and by changes in morphology [1]. Changes of platelet morphology, including loss of discoid shape and degranulation or vacuolization, which are seen with platelet activation, can occur during storage and lead to impaired functions after transfusion [2]. With the introduction of acetate-containing additive solutions [3,4], the storage conditions of platelets derived from pooled bu€y coats and apheresis were improved. However, the success of platelets stored in acetate-containing media is necessary with a high rate, at least 15±20%, of autologous plasma carryover. This issue with plasma carryover, which partly provides glucose for fuel [5], is not well de®ned. With a lower plasma portion, the remaining bu€er capacity and glucose levels are not able to allow successful storage of platelets [6], and the requirements for the improved plasma-free synthetic medium become very rigorous [7]. In this study, we evaluated the in vitro functions and in vivo survival of platelets preserved in newly developed synthetic media, named Fukushima Cocktails (FCs), containing glycerol at di€erent concentrations. 2. Materials and methods 1. Studies of long-term preservation of platelets for determining the suitable concentrations of glycerol. The compositions of FC-A, FC-B and FC-C and Control-1 medium without glycerol are shown in Table 1. PCs were prepared from 5000 ml of whole blood by plateletpheresis with a CS-3000 cell separator (Baxter Healthcare Corporation, IL, USA).

Table 1 Compositions (mM) of FCs and a control solution NaCl KCl MgCl2 Na2 HPO4 Na acetate Na3 citrate Citric acid Glucose Glycerol pH Osmolarity (mOsm)

FC-A

FC-B

FC-C

90 5 3 33 25 15 2 25 25 7.4 400

90 5 3 33 25 15 2 25 50 7.4 430

65 5 3 33 25 15 2 25 100 7.4 430

Control-1

±

90 5 3 33 25 15 2 50

7.4 400

Two preparations of platelets of the same ABO blood type were each mixed with 200 ml of plasma and put into ®ve polyole®n storage bags (KBP-PO, Kawasumi Lab., Japan). After centrifugation, the plasma was removed from each bag and 100 ml of synthetic medium (FC-A, B, C or Control-1) or autologous plasma (Control-2) was added. Except for the plasma control, there was a residue of approximately 5 ml (5%) of autologous plasma in each bag. The platelets were stored for up to 9 days at 22°C with agitation at 50±60 strokes/min on a ¯at shaker. Platelet functions were tested after 5, 7 and 9 days of storage. Platelet counts and mean platelet volumes (MPV) were determined using an electronic cell counter (Sysmex K-2000, TOA, Japan). Hypotonic shock response (%HSR) and aggregation were measured after diluting the platelets to 2:5  108 /ml using autologous plasma. %HSR was determined using a photometer at 610 nm (UVIDEC40, JASCO, Japan) and was expressed as the decrement in light transmission during recovery from shock as a percentage of the total increment, as described elsewhere [8]. Platelet aggregation response to double stimuli, 10 lM ADP and 10 lg/ml collagen (®nal concentrations), was measured with a lumiaggregometer (P.I.C.A. C660, Chronolog, USA). The aggregation response was expressed as the maximum change in transmittance after stimulation compared to platelet-poor plasma. The pH and HCO3 levels of supernatants of PCs were measured using a pH/blood gas analyzer (ABL300, Radiometer, Copenhagen, Denmark).

T. Yuasa et al. / Transfusion Science 23 (2000) 37±46

To evaluate spontaneous activation of platelets in storage, levels of b-thromboglobulin (b-TG) and thromboxane B2 (TXB2 ) were measured by the enzyme-linked immunosorbent assay and radio-isotope assay, respectively. Lactate and ATP levels were measured by enzymatic assays. Platelet morphology was examined using Nomarski interference contrast light microscopy and was expressed as the percentage of discoid cells. The swirling patterns of preserved platelets were observed macroscopically and given one of three grades (++, +, or )) [9]. 2. Study of the relation between plasma-exchanged rate and platelet functions. FC-B including 50 mM glycerol was examined as follows. The functions and the viability of platelets preserved in three di€erent plasma exchange rates for 7 days were evaluated. We made three di€erent plasmaexchange rates of PCs; FC80, FC90 and FC99 (80%, 90%, and 99%) and plasma-control PCs. Platelets prepared from two donors with the same ABO blood type were divided into four bags (100 ml of each), centrifuged and 80, 90 ml or most of 100 ml of platelet-poor plasma was removed and replaced with the same volume of FC-B or autologous plasma. The plasma-exchange rate was calculated from the concentrations of total protein (TP; g/dl) in the ¯uid phase. MPV, %HSR, aggregation, pH and swirling pattern were measured on days 1, 3, 5 and 7. 3. In vivo survival of platelets stored in FC-B. In vivo survival was studied using platelets obtained from four healthy donors by apheresis and stored for 3 days in FC-B or in autologous plasma as a control. The platelets stored in FC or autologous plasma from two donors were labeled with 51 Cr, or 111 In, respectively, as described elsewhere [10]. Platelets from the other two donors were labeled vice versa; the platelets in FC-B were labeled with 111 In and those in autologous plasma with 51 Cr. Labeled platelets were reinfused into the original donors in 5 min. Five ml of blood samples were drawn before and 15 min at the basic line (estimated 100% recovery of transfused platelets), 24 and 48 h after reinfusion. Platelet-rich plasma (PRP) was obtained by centrifugation of blood samples at 690 g

39

for 5 min and measuring the numbers of platelets. One ml of PRP was assayed for radioactivity levels using a two channel gamma counter (ARC-351, ALOKA, Japan). One window was set for the 51 Cr peak and the other for the 111 In peak. Twentyfour- and 48-hour post-transfusion survivals were calculated from the activities per 106 platelets in PRP as a percentage of the 15 min activities after subtracting pre-transfusion from post-transfusion radioactivity. 4. Safety and corrected count increment of the transfusion of 3-day platelets stored in FC-B. Using three healthy donor-recipients, the safety of transfusion of 1  1011 platelets stored for 3 days in 100 ml of FC-B was evaluated by measuring blood pressure, heart rate, body temperature, blood cell counts and doing serologic and biochemical tests (total protein, albumin, bilirubin, total cholesterol, urea nitrogen, creatinine, uric acid, AST, ALT, LDH, ALP and c-GTP) before and after 1, 3 h, 1 and 2 days of transfusion. In addition, to evaluate the in vivo survival of infused platelets in these experiments, the corrected count increment (CCI) 1 h after transfusion was measured using the following equation: CCI ˆ …Platelet countpost ÿ Platelet countpre †  BSA…m2 † : Number of platelets transfused …multiples of 1011 † When CCI at 1 h after transfusion was above 7000±10,000/ll, platelet transfusion was judged to be successful [11].

3. Ethics Committee approval The Institutional Ethics Committee gave approval for the research. A written informed consent was obtained from every volunteer.

4. Statistical analysis Data are expressed as mean  standard deviation (S.D.), and were analyzed using the paired and unpaired StudentÕs t-test. A di€erence of p < 0:05 was considered signi®cant statistically.

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T. Yuasa et al. / Transfusion Science 23 (2000) 37±46

5. Results 1. Functions of platelets preserved in FC-A, FC-B and FC-C (Table 2). Platelet counts did not di€er signi®cantly among the ®ve groups during 9 days of storage. The MPV of Control-1 (lacking glycerol) increased at day 9 signi®cantly …P ˆ 0:02†. %HSR of platelets in all the groups decreased after 9 days of storage. However, there were no signi®cant differences between the three FCs and the plasma control at day 5, and a control lacking glycerol was signi®cantly lower than FC-B and the plasma control at day 7. Although %HSR of FC-B and the plasma control seemed to be superior to FC-A, FCC or Control-1 without glycerol at days 7 and 9, there were no signi®cant di€erences between them. The aggregation of platelets stored in FC-A, FC-B and FC-C induced by double stimuli, ADP and collagen, did not di€er from platelets stored in plasma but was superior to control medium lacking glycerol. The pH decreased in the three FCs and the two controls from day 5 to day 9. The HCOÿ 3 levels in all samples decreased from day 5 to day 9, and the levels in FCs and Control-1 were lower than in the plasma control during storage. b-TG in the supernatants of all ®ve PCs increased during the 9 days of storage. There were no signi®cant di€erences between any of the samples on days 5 and 7. However, on day 9, Control1 lacking glycerol, had a higher level of b-TG with signi®cant di€erence from FC-A, FC-B and the plasma control. TXB2 levels in the supernatants increased during 9 days of storage in all ®ve groups. Lactate was increased in the supernatants of every group with similar kinetics. Platelet ATP levels in FC-B and FC-C were similar to those of the plasma control at days 5 and 7. Those of FC-A containing 25 mM glycerol and Control-1 without glycerol were less than 2.5 mg/dl (the limit of detection) and were signi®cantly lower than the other three groups until 7 days. However, platelet ATP levels in all four synthetic media were less than 2.5 mg/dl at day 9. The percentage of platelets with discoid morphology decreased gradually during storage in all ®ve groups.

Swirling pattern was clearly observed in all samples on day 5, but not on days 7 and 9, with platelets stored in FC-C and in Control-1 lacking glycerol. 2. Study of the relation between plasma exchange rates and platelet functions. Functions of platelets stored for 7 days in FC-B are shown in Table 3. The actual plasma-exchange rates estimated by the media protein levels of FC80, FC90 and FC99 were 84.3%, 93.6% and 98.3%, respectively. Platelet functions stored in the media were well maintained until 5 days of storage, but platelets stored in FC99 showed poorer values of pH and %HSR and swirling pattern on days 7 compared to the control (p ˆ 0:005 in pH, p ˆ 0:04 in %HSR). 3. In vivo survival of platelets stored for 3 days in FC-B. The plasma-exchanged rates of PCs prepared with FC-B in this study were 90±95%. As shown in Fig. 1, the mean 24-hour survival rate of platelets preserved for 3 days in FC-B (76:8  10:1%, mean  S:D:) and autologous plasma (74:9  7:9%), were both excellent. The 48hour post-transfusion survival rate of platelets stored in FC-B …59:7  11:8%† was not di€erent from platelets stored in autologous plasma …56:9  4:5%†. 4. Safety and CCI of the transfusion of platelets stored for 3 days in FC-B. Vital signs, blood cell counts and serologic biochemical parameters were monitored before and after transfusion of autologous platelets which were preserved for 3 days. The summaries of results are shown in Tables 4 and 5. There were no adverse e€ects from the transfusion of the platelets until 3 days after transfusion (data partly shown). The CCI 1 h after transfusion of PCs in FC-B was 20; 300  2100=ll, showing that the transfused platelets were kept in good condition for 3 days. 6. Discussion Non-hemolytic reactions (NHR) after platelet transfusion vary from urticarial to severe anaphylactic reactions and occur with a frequency of 1±2% [12]. Most of the NHR after transfusion of platelet concentrates are allergic (51%), followed by febrile reactions (32%), and circulatory distress

T. Yuasa et al. / Transfusion Science 23 (2000) 37±46

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Table 2 Platelet functions and morphology and levels of biochemicals during 9-day storage (n ˆ 3 (mean ‹ 1 S.D.)) Days

FC-A

FC-B

FC-C

Control-1 without glycerol

Control-2 (plasma)

Platelet count (109 /l)

5 7 9

1150 ‹ 60 1120 ‹ 61 1023 ‹ 68

1137 ‹ 60 1052 ‹ 69 987 ‹ 35

1103 ‹ 71 1067 ‹ 21 970 ‹ 44

1173 ‹ 95 1123 ‹ 95 977 ‹ 45

1177 ‹ 92 1247 ‹ 90 1093 ‹ 76

MPV (¯)

5

7.8 ‹ 0.1

7.8 ‹ 0.2

8.5 ‹ 0.2

8.4 ‹ 0.31

7.1 ‹ 0.1

7

8.1 ‹ 0.3

8.4 ‹ 0.3

8.2 ‹ 0.2

8.9 ‹ 0.4

7.3 ‹ 0.3

9

8.0 ‹ 0.22

8.3 ‹ 0.53

8.5 ‹ 0.44

9.6 ‹ 0.55

7.4 ‹ 0.26

5

73.7 ‹ 8.5

68.2 ‹ 3.7

68.1 ‹ 2.2

57.3 ‹ 3.11

70.6 ‹ 2.92

7

60.2 ‹ 4.9

68.3 ‹ 3.83

61.3 ‹ 4.6

49.5 ‹ 5.44

69.1 ‹ 5.45

9

29.6 ‹ 1.46

35.1 ‹ 5.67

26.8 ‹ 7.28

31.8 ‹ 6.29

37.3 ‹ 4.910

Aggregation (%) ADP (10 lM) + Collagen (10 lg/ml)

5 7 9

73.9 ‹ 8.5 57.7 ‹ 6.8 47.4 ‹ 6.0

77.0 ‹ 7.0 65.7 ‹ 4.0 55.7 ‹ 5.9

75.6 ‹ 6.5 63.3 ‹ 11.0 40.0 ‹ 6.1

61.0 ‹ 9.6 55.3 ‹ 7.2 33.0 ‹ 5.7

78.0 ‹ 8.7 68.0 ‹ 7.2 48.7 ‹ 5.1

pH

5 7 9

6.93 ‹ 0.03 6.78 ‹ 0.03 6.75 ‹ 0.06

6.85 ‹ 0.07 6.69 ‹ 0.13 6.45 ‹ 0.26

6.97 ‹ 0.03 6.88 ‹ 0.05 6.76 ‹ 0.09

6.94 ‹ 0.02 6.91 ‹ 0.04 6.77 ‹ 0.14

7.18 ‹ 0.01 7.03 ‹ 0.07 6.95 ‹ 0.08

HCOÿ 3 mM/l

5 7 9

3.9 ‹ 0.1 3.0 ‹ 0.1 1.7 ‹ 0.4

3.9 ‹ 0.6 2.6 ‹ 1.0 1.6 ‹ 1.0

3.8 ‹ 0.2 2.4 ‹ 0.7 1.2 ‹ 1.0

4.0 ‹ 0.2 2.8 ‹ 0.8 1.7 ‹ 1.1

11.3 ‹ 0.4 9.0 ‹ 0.4 5.9 ‹ 1.3

b-TG (lg/ml)

5

13.0 ‹ 3.0

11.9 ‹ 1.0

10.5 ‹ 0.8

11.9 ‹ 1.5

12.2 ‹ 1.0

7 9

13.3 ‹ 2.3 15.1 ‹ 0.11

13.5 ‹ 0.2 14.4 ‹ 0.52

13.8 ‹ 1.8 17.5 ‹ 3.33

18.7 ‹ 5.5 26.0 ‹ 4.54

14.5 ‹ 0.9 17.0 ‹ 1.45

TXB2 (ng/ml)

5 7 9

3.7 ‹ 0.8 6.3 ‹ 0.8 11.5 ‹ 1.9

2.9 ‹ 1.4 4.2 ‹ 0.8 9.7 ‹ 1.4

3.3 ‹ 0.9 6.7 ‹ 1.6 10.8 ‹ 1.5

3.5 ‹ 0.7 7.9 ‹ 1.8 15.6 ‹ 4.1

3.2 ‹ 0.8 5.1 ‹ 0.5 12.5 ‹ 1.6

Lactate (mg/ml)

5

57.4 ‹ 2.1

65.3 ‹ 11.2

53.4 ‹ 2.31

46.9 ‹ 8.3

79.3 ‹ 8.12

7 9

87.9 ‹ 6.3 124.5 ‹ 5.8

93.6 ‹ 20.2 101.8 ‹ 22.1

80.7 ‹ 10.23 120.2 ‹ 15.8

94.3 ‹ 20.6 119.8 ‹ 12.8

101.9 ‹ 15.44 143.7 ‹ 27.5

5 7 9

<2.5 <2.5 <2.5

2.9 ‹ 0.1 2.7 ‹ 0.2 <2.5

2.7 ‹ 0.3 2.8 ‹ 0.2 <2.5

<2.5 <2.5 <2.5

3.2 ‹ 0.2 3.0 ‹ 0.5 2.7 ‹ 0.3

HSR (%)

ATP (mg/dl)

P-value by t-test

*1 vs *5; P ˆ 0.02 *2 vs *6; P ˆ 0.005 *3 vs *6; P ˆ 0.03 *4 vs *6; P ˆ 0.01 *5 vs *6; P ˆ 0.002 *1 vs *2; P ˆ 0.002 *3 vs *4; P ˆ 0.008 *4 vs *5; P ˆ 0.02 *6 to *9 vs *10; NSa

*1 vs *4; P ˆ 0.02 *2 vs *4; P ˆ 0.01 *3 vs *4; NS *4 vs *5; P ˆ 0.03

*1 vs *2; P ˆ 0.006 *3 vs *4; NS

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T. Yuasa et al. / Transfusion Science 23 (2000) 37±46

Table 2 (Continued)

a b

Days

FC-A

FC-B

FC-C

Control-1 without glycerol

Control-2 (plasma)

Morphology (discoid %)

5 7 9

28 ‹ 4 22 ‹ 5 12 ‹ 5

28 ‹ 6 21 ‹ 5 14 ‹ 4

26 ‹ 5 20 ‹ 3 10 ‹ 5

20 ‹ 6 15 ‹ 6 10 ‹ 7

22 ‹ 3 20 ‹ 4 15 ‹ 6

Swirling pattern testb

5 7 9

++ ++ +

++ ++ +

++ + ±

++ + ±

++ ++ +

P-value by t-test

NS: Not signi®cant. ++ Denotes excellent; + good; ± poor.

Table 3 Platelet functions during 7-day storage in FC-B (n ˆ 3 (mean  1 S.D.)) Planned plasma-exchanged rate 80%

90%

99%

Control (plasma)

84.3 ‹ 5.9

93.6 ‹ 1.1

98.3 ‹ 0.1

±

Days 1 3 5 7

1174 ‹ 119 1134 ‹ 132 1115 ‹ 101 1083 ‹ 99

1267 ‹ 49 1236 ‹ 99 1196 ‹ 81 1173 ‹ 102

1128 ‹ 36 1118 ‹ 17 1086 ‹ 32 1031 ‹ 101

1136 ‹ 58 1104 ‹ 85 1086 ‹ 101 1041 ‹ 89

MPV (¯)

1 3 5 7

7.6 ‹ 1.0 7.5 ‹ 0.9 7.5 ‹ 0.8 7.8 ‹ 1.1

7.6 ‹ 0.9 7.4 ‹ 0.7 7.4 ‹ 0.8 7.9 ‹ 1.0

7.8 ‹ 1.0 7.9 ‹ 1.1 8.6 ‹ 0.9 10.0 ‹ 1.51

7.1 ‹ 0.7 6.9 ‹ 0.7 6.9 ‹ 0.7 7.0 ‹ 0.72

HSR (%)

1 3 5 7

66.9 ‹ 1.7 68.6 ‹ 5.3 60.2 ‹ 4.0 54.0 ‹ 2.7

67.6 ‹ 9.1 66.9 ‹ 8.4 58.8 ‹ 11.5 54.0 ‹ 13.2

63.6 ‹ 2.9 61.1 ‹ 0.6 52.7 ‹ 4.2 46.1 ‹ 2.71

73.0 ‹ 8.4 69.1 ‹ 10.9 67.4 ‹ 8.3 59.8 ‹ 7.22

Aggregation (%) ADP (10 lM) + Collagen (10 lg/ml)

1 3 5 7

89.6 ‹ 18.0 85.8 ‹ 20.4 66.8 ‹ 6.6 65.9 ‹ 8.1

88.7 ‹ 19.6 84.0 ‹ 27.7 74.3 ‹ 2.1 65.7 ‹ 21.0

93.3 ‹ 11.5 87.3 ‹ 21.9 68.7 ‹ 10.7 53.7 ‹ 12.1

87.7 ‹ 21.4 85.3 ‹ 25.4 66.7 ‹ 8.5 54.7 ‹ 4.5

pH

1 3 5 7

7.07 ‹ 0.08 6.99 ‹ 0.09 6.98 ‹ 0.13 6.87 ‹ 0.14

7.06 ‹ 0.06 6.97 ‹ 0.04 6.91 ‹ 0.07 6.75 ‹ 0.12

7.05 ‹ 0.03 6.90 ‹ 0.04 6.74 ‹ 0.081 6.49 ‹ 0.123

7.18 ‹ 0.10 7.08 ‹ 0.10 7.04 ‹ 0.062 6.95 ‹ 0.084

Swirling pattern testa

1 3 5 7

++ ++ ++ +

++ ++ ++ +

++ ++ ++ )

++ ++ ++ +

Actual plasma-exchanged rate with FC (%) Platelet count (109 /l)

a

P-value by t-test

*1 vs *2; P ˆ 0.01

*1 vs *2; P ˆ 0.04

*1 vs *2; P ˆ 0.01 *3 vs *4; P ˆ 0.005

++ Denotes excellent; + good; ± poor.

(17%) [13]. Replacing plasma with a synthetic medium could minimize allergic and circulatory reactions, which are induced by immune responses to allo-plasma proteins.

Some investigators have reported synthetic media or additive solutions for platelet storage at room temperature (22°C) [14±17], at 4°C [18] or at )80°C [19±23]. Except for platelets preserved at

T. Yuasa et al. / Transfusion Science 23 (2000) 37±46

43

Fig. 1. In vivo survival (%) of transfused platelets, preserved in FC-B or in autologous plasma for 3 days (n ˆ 4) (N.S.: not signi®cant).

Table 4 Vital signs and blood counts before and after transfusion of FC-PC (n ˆ 3 (mean  1 S.D.)) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Pulse (/min) Temperature (°C) White blood cells (103 /ll) Red blood cells (106 /ll) Hemoglobin (g/dl) Hematocrit (%) Mean corpuscular volume (¯) Platelets (103 /ll)

Before

1 h after

1 day after

139 ‹ 7 73 ‹ 2 78 ‹ 9 36.0 ‹ 0.4 5.7 ‹ 0.8 5.10 ‹ 0.06 15.6 ‹ 0.7 45.7 ‹ 0.7 89.6 ‹ 2.0 184.0 ‹ 32.7

134 ‹ 13 78 ‹ 4 69 ‹ 3 36.1 ‹ 0.4 6.0 ‹ 0.2 5.08 ‹ 0.04 15.5 ‹ 0.8 45.3 ‹ 0.7 89.1 ‹ 2.1 194.0 ‹ 32.0

126 ‹ 11 68 ‹ 7 73 ‹ 4 36.1 ‹ 0.3 6.0 ‹ 0.8 5.11 ‹ 0.16 15.6 ‹ 1.2 45.6 ‹ 2.7 89.3 ‹ 3.1 215.7 ‹ 35.2

room temperature they lose their discoid shape by cold activation and show signi®cantly lower posttransfusion recovery than those preserved at room temperature. One of the glucose-free additive solutions, originally designated PAS [17,24] which contains sodium/potassium chloride, citrate, phosphate and mannitol was recently improved by the addition of acetate (PAS2) and was found to allow storage of PCs for several days with good results [25,26]. For glucose-free media, the mini-

mal residual plasma carryover necessary for storage of platelets for 5 days, has already been shown to be in the range of 10±20% [27] or 30% [28]. The amount of glucose included in medium with 15± 20% plasma carryover could provide enough energy from glycolysis during 5 days of storage [29]. Shimizu et al. [4] pointed out that successful 5day storage of platelets was achieved in a synthetic medium, termed Seto Solution, which contains NaCl, KCl, MgCl2 , NaH2 PO4 , Na2 HPO4 , Na

44

T. Yuasa et al. / Transfusion Science 23 (2000) 37±46

Table 5 Biochemical tests before and after transfusion of autologous platelets stored in FC-B (n ˆ 3 (mean  1 S.D.)) Total protein (g/dl) Albumin (mg/dl) Direct bilirubin (mg/dl) Indirect bilirubin (mg/dl) Total cholesterol (mg/dl) BUN (mg/dl) Creatinine (mg/dl) Uric acid (mg/dl) AST (IU/L/37°C) ALT (IU/L/37°C) LDH (IU/L/37°C) ALP (IU/L/37°C) c-GTP (IU/L/37°C)

Before

1 h after

1 day after

7.3 ‹ 0.2 4447 ‹ 121 0.2 ‹ 0.0 0.4 ‹ 0.2 191 ‹ 6 13.3 ‹ 2.4 0.8 ‹ 0.1 5.6 ‹ 0.2 19 ‹ 5 19 ‹ 7 270 ‹ 42 192 ‹ 34 28 ‹ 10

7.2 ‹ 0.4 4357 ‹ 154 0.2 ‹ 0.1 0.4 ‹ 0.1 192 ‹ 10 13.1 ‹ 2.1 0.8 ‹ 0.1 5.6 ‹ 0.2 18 ‹ 4 19 ‹ 5 261 ‹ 58 186 ‹ 30 28 ‹ 8

7.4 ‹ 0.5 4350 ‹ 376 0.3 ‹ 0.2 0.3 ‹ 0.0 195 ‹ 11 14.7 ‹ 1.3 0.8 ‹ 0.1 6.3 ‹ 0.5 21 ‹ 6 20 ‹ 7 286 ‹ 86 197 ‹ 34 29 ‹ 7

acetate, Na3 citrate, maltose and glucose. Acetate is eciently metabolized to CO2 by platelets and prolongs the period of e€ective storage compared to acetate-free, non-plasma media [30]. Because acetate is metabolized in the citric acid cycle, it can provide the energy needs of platelets with the production of much less acid than when glucose serves as the substrate for the glycolytic pathway. The PCs prepared with Seto Solution consist of 11% plasma carryover [31], which might be essential for the storage of platelets for longer than 3 days. We con®rmed that the bicarbonate (HCOÿ 3) values were 5:1  0:9 mM/l at day 1 and 5:2  0:7 mM/l at day 3. Unfortunately, we did not evaluate the day 0 value. But it can be expected that the bicarbonate concentration was very low initially, because FCs do not contain bicarbonate. This phenomenon can be explained by the reason why bicarbonate is produced by acetate metabolism [6]. We anticipated the bene®cial e€ects of glycerol for platelet functions, as glycerol is known to protect red cells from damage caused by freezing [32]. When platelets were stored in a synthetic medium lacking glycerol, their functions in this study were inferior to those stored in plasma or media containing glycerol. There was decreased %HSR, less aggregation, less swirling pattern and increased MPV. Generally, platelets stored for long periods in liquid show morphological changes with decrease in the percentage of discoid cells and increase in MPV. Morphological changes of platelets are known to adversely a€ect post-trans-

fusion platelet survival [33] and re¯ect a decrease in their amounts of ATP and ADP [34]. Decreased ATP levels re¯ect a decrease in %HSR. In this regard, FC-B with 50 mM glycerol was superior to Control-1 without glycerol. On the other hand, when b-TG is released from a-granules in platelets, the TXB2 level in plasma increases. Measurements of platelet activation markers (aggregation, b-TG, TXB2 ) showed that FC-A and FC-B were as effective in preserving platelets as regular PCs in plasma. Therefore, it is suggested that FCs do not activate platelets spontaneously during storage for 7 days. By adding glycerol to acetate-containing solution, the storage of platelets can be greatly improved even in as small as 5±6% plasma carryover. Although glycerol confers protection on the lipid lea¯et or membrane proteins of red blood cells under high hydrostatic pressures [35], the mechanism of bene®cial e€ect on platelet preservation is not known. Among a lot of structural, biochemical and functional alterations that lead to an impaired function after transfusion, swollen open canalicular system (OCS) may be a critical damage which induces changes in the structure of a-granules, and decrease in the reactivity of stored platelets to weak agonists [1]. Therefore, it is tempting to speculate the glycerol e€ect that the protection from the decrease of a-granular contents by delaying the dissolve a-granules within OCS. This hypothesis is supported by the observation that in bu€y coat-derived platelets a constant a-granular

T. Yuasa et al. / Transfusion Science 23 (2000) 37±46

content was found whereas the decreased a-granular content was observed in association with the increased presence of OCS after storage [1]. The substances of a-granules are endocytosed from the blood plasma, into which they are secreted by various white blood cells, hepatocytes, endothelial cells and other cells [36]. The endocytosis of substances is likely another reason, in addition to the source of glucose, that previous synthetic solutions for platelet preservation are necessary for 10±30% plasma carryover to maintain platelet functions for 5 days. The actin ®laments of platelets are connected with glycoproteins of the a-granular membranes and with spectrin from the membrane skeleton that underlie the lipid bilayer of the plasma membrane [37]. Taking part in baroprotective action on red cell membrane [35], glycerol may act as a membrane stabilizer on platelets. Thirdly, we have to mention about the released substances, such as ADP, which activate the remaining platelets [38]; glycerol might act as an antagonist of the ADP receptor in the storage medium like other nucleotides such as AMP and ATP [39]. From these considerations, we conclude that glycerol is bene®cial for extending the preservation period of platelets at 22±26°C and maintaining them in good condition, and FC-B containing 50 mM glycerol allows the removal of most of the plasma (94%) from PCs. Therefore, the frequency of allergic reactions after transfusion of platelets of plasma by using FC should be less than those with other arti®cial media. A concentration of around 50 mM glycerol is bene®cial for more than 7 days of liquid storage when plasma was depleted and replaced with FC. Functions and viability markers of platelets stored in FC with 2% residual plasma were as good as those stored in plasma for 3 days. These in vitro data encouraged us to evaluate in vivo survival. Our in vivo 24- and 48-hour survival studies con®rmed that platelets stored for 3 days in FC-B are similar to platelets stored in autologous plasma. Also, the CCI calculated by a platelet count increment at 1-hour post-transfusion was satisfactory [40]. The safety of the transfusion of FC-PCs stored for 3 days was also con®rmed; none of the four

45

recipients had any adverse e€ects related to the transfusion of FC-PCs. In summary, the use of FC containing 50 mM glycerol for platelet storage caused no particular problems in our studies, and had in fact several advantages in terms of successful platelet transfusion.

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