Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution

Cryobiology 62 (2011) 100–106

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Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution q L.N. Johnson ⇑, K.M. Winter, S. Reid, T. Hartkopf-Theis, D.C. Marks Research and Business Development, Australian Red Cross Blood Service, Sydney, Australia

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Article history: Received 16 November 2010 Accepted 10 January 2011 Available online 15 January 2011 Keywords: Cryopreservation Dimethyl sulfoxide Platelet Platelet additive solution SSP+

a b s t r a c t Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me2SO) and stored for extended periods at 80 °C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me2SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at 80 °C. The cryopreserved platelet units (n = 9) were rapidly thawed at 37 °C, reconstituted in 50% SSP+/plasma and stored at 22 °C. Platelet recovery and quality were examined 1 and 24 h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24 h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me2SO and additive solution produces acceptable in vitro platelet quality. Ó 2011 Elsevier Inc. All rights reserved.

Introduction Platelet concentrates produced from buffy-coats or apheresis can be stored for 5 days at room temperature [8,17,25]. However, when platelet concentrates are cryopreserved, they can be stored for several years [24,41]. If platelets could be effectively cryopreserved then wastage in times of surplus could be minimized, leading to more effective inventory and supply chain management. In addition, a bank of HLA-, HPA-typed and IgA-deficient platelet products could be prepared, further improving inventory management of these rare platelet types. There is also an unmet need in remote locations for suitable blood products. This usually occurs in cases of trauma where maintaining fresh products is not feasible given their short shelf-life and the distance from Australia [18]. Therefore, a frozen stock of blood products would negate these problems.

Abbreviations: Me2SO, dimethyl sulfoxide; PAS, platelet additive solution; HSR, hypotonic shock response; ADP, adenosine diphosphate; PS, phosphatidylserine; Dw, mitochondrial membrane potential; TRALI, transfusion related acute lung injury. q Statement of funding: This work was funded by grants from the Australian Red Cross Blood Service and the Australian Defence Force. Neither funding source played any role in the collection, analysis, and interpretation of data or in the writing of the report or the decision to submit the paper for publication. ⇑ Corresponding author. Address: Research and Business Development, The Australian Red Cross Blood Service, 153 Clarence Street Sydney, NSW 2000, Australia. E-mail address: [email protected] (L.N. Johnson).

0011-2240/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2011.01.003

Platelet cryopreservation is not carried out routinely in blood banks due to the cumbersome and expensive nature of the currently available methods. Further, several methods for cryopreservation have been described, with variable success [26]. However, cryopreserved platelets have been shown to be suitable for transfusion, and may even be more hemostatically active than liquidstored platelets [24,28,32]. The effectiveness of several cryoprotectants for platelet freeze/ thawing has been examined. These include dimethyl sulfoxide (Me2SO), trehalose, propylene glycol, ethylene glycol, glycerol, glycerol–glucose, nitric oxide and dextran-40 [2,10,27,30,37]. However, Me2SO at high concentrations (5–6%) is currently considered the best cryoprotectant for platelets [25]. Due to the toxic effects of this compound, pre-transfusion washing is required, which may result in platelet loss and increased platelet activation [32]. Alternatively, the platelet product can be washed prior to freezing, which simplifies the procedure and improves product quality [39]. However, the majority of these studies have been carried out with apheresis-derived platelets, stored and frozen in plasma. In Australia, buffy-coat-derived platelets are prepared in plasma and a platelet additive solution (PAS), known as SSP+ or PAS-IIIM. PAS can be substituted for plasma as it has a more standardized composition than plasma; it conserves plasma resources; and can decrease plasma-related adverse reactions, such as transfusionrelated acute lung injury (TRALI) [34,42]. The substitution of plasma with an optimal PAS may also improve platelet viability and

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hemostatic function, thereby allowing for prolongation of platelet storage [14,19]. Further, the use of PAS for cryopreservation may also provide the potential to pre-prepare the cryoprotectant solution. As such, we wanted to determine whether this plasmareduced product could be frozen, without loss of in vitro platelet quality. A single study has evaluated the benefit of freezing platelets in PAS, with different ratios of plasma carryover [9]. However, Composol was used in this study which has a slightly different composition to SSP+. The purpose of the current study was to determine whether freezing and thawing platelets in 6% Me2SO and SSP+, in place of plasma, would result in a high quality cryopreserved platelet product, when compared to standard liquid-stored platelets prepared in SSP+.

Materials and methods Preparation of platelet concentrates For platelet preparation, four buffy-coats were pooled with 300 mL SSP+ (Macopharma, France) and separated by centrifugation (1350 rpm, 6 min) and extraction using a semi-automated blood extractor (Optipress II; Baxter Healthcare, IL, USA). The final platelet concentrates were leukoreduced by filtration using Imuguard III-S PL filters and maintained in the associated platelet storage bag (Terumo, NJ, USA). Cryopreservation of platelet concentrates Platelets were frozen using the method described by Hornsey and coworkers [23], with several modifications. Buffy-coat-derived pooled platelet concentrates (n = 9) were produced in 70% SSP+/ plasma, as described above, and stored at 22 °C with agitation. On day 2 of storage, the platelets were transferred to a 500 mL cryopreservation bag (CryoMACS; Miltenyi Biotec, Germany), using a sterile connection device (TSCD-II; Terumo, NJ, USA). A control sample was taken prior to freezing (pre-freeze sample), by collection of 5 mL into the sampling pouch of the original platelet storage bag. Sterility was maintained by the use of filtered Me2SO (Hybri-Max™; Sigma, MO, USA), sterile syringes, solutions and tubes, and all procedures were carried out at room temperature (20–22 °C) in a laminar flow hood. A mixture of 50% Me2SO/plasma (75 mL) was added to the platelet concentrate, via a syringe, at a rate of 10 mL per minute with agitation, to give a final concentration of 6% Me2SO. The platelets were pelleted by centrifugation (2000g for 10 min) and the supernatant removed using an Optipress II, leaving approximately 26 mL. The platelets were gently resuspended in the remaining supernatant. The platelet product was placed inside an ethylene vinyl acetate (EVA) overwrap bag (CryoMACS; Miltenyi Biotec, Germany) and a metal freezing cassette (Thermo Fisher Scientific Inc., MA, USA) and frozen by placing directly into a 80 °C freezer. For rapid thawing, the cryopreserved platelet concentrates were incubated at 37 °C in a water bath. The thawed platelet unit was sterile-docked to a platelet storage bag (ELP; CaridianBCT, CO, USA) containing 300 mL 50% plasma/SSP+. AB negative plasma was used, to ensure a universally transfusable product. The platelets were gently transferred to the storage bag and mixed to reconstitute. Platelet concentrates were subsequently stored at 22 °C with agitation. Platelet recovery was examined 1 and 24 h postthaw and compared to the pre-freeze sample. Laboratory analysis of platelet concentrates Platelet recovery was determined by evaluating platelet count and mean platelet volume (MPV) on a CellDyn 3200 hematology

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analyzer (Abbott Diagnostics, Sydney, Australia). The platelet swirl was characterized by visual assessment. Platelet viability was determined using two fluorescent dyes, Calcein-AM and FM4-64, as previously described [17]. Calcein-AMbright/ FM4-64dim cells are viable, while the opposite staining is indicative of dead cells. The cells were analyzed by flow cytometry (FACSCanto II, Becton Dickinson, NJ, USA) within 1 h of staining. Assessment of pH, lactate, glucose, bicarbonate, pO2 and pCO2 levels were carried out using the iSTAT blood gas analyzer in conjunction with the CG4+ and glucose cartridges (Abbott Diagnostics, Sydney, Australia). Hypotonic shock response (HSR) was measured using a platelet aggregometer (Helena Laboratories, TX, USA). Briefly, platelet concentrates were diluted in plasma to 300,000/lL. The change in light transmission in response to phosphate buffer solution and Milli-Q water and was monitored for 4 min. The HSR percentage (% recovery) was calculated according to previously described methods [21]. For measurement of phosphatidylserine (PS) exposure, platelets (1  106 cells) were stained with 5 lL of annexin-V-FITC in binding buffer (Biolegend, CA, USA) for 15 min. The cells were then diluted with annexin-V binding buffer and analyzed within 1 h by flow cytometry. Changes in platelet mitochondrial transmembrane potential (Dw) were determined using JC-1 dye (Biotium Inc., CA, USA). The method of Albanyan and colleagues [1] was adapted with minor modifications. Platelet glycoproteins were assessed by staining platelets (3  106) in Tyrode’s buffer for 20 min at room temperature with the following antibodies: anti-human CD62P-PE (BD Biosciences, CA, USA), anti-human CD63-PE (Biolegend), anti-human CD41a-FITC (BD Biosciences), anti-human CD61-FITC (Dako, Glostrup, Denmark), anti-human CD42b-PE (BD Biosciences) and antihuman CD47-PE (Biolegend). Anti-IgG1j-PE and anti-IgG1j-FITC were used as isotype controls (Biolegend). Samples were then washed, fixed and analyzed by flow cytometry. Cytokines were measured from the supernatants of platelet concentrates. Supernatants were prepared by double centrifugation at 1600g for 20 min then 12,000g for 5 min. Supernatants were collected and stored at 80 °C until assayed. Commercially available ELISA kits were used according to the manufacturer’s instructions (R&D Systems Inc., MN, USA for CD40L, RANTES, sCD62P, PDGF-AB, PF4, TGF-b1, EGF and Biolegend for IL-1b and TNF-a). Glycocalicin ELISAs were carried out according to previously published methods [19]. All samples were tested in triplicate. Turbidometric aggregation was performed with 225 lL of platelet rich plasma (300,000/lL) in response to 20 lM adenosine diphosphate (ADP; Sigma, MO, USA) or 10 lg/mL collagen (Helena Laboratories) using an aggregometer (Helena Laboratories). Aggregation was assessed as maximal aggregation (% Max) at 5 min after adding the agonist. Statistical analysis Results are expressed as mean ± standard deviation (SD). Results obtained prior to freezing and at 1 and 24 h post-thawing were compared, using a two-sided paired t-test. A p-value of less than 0.05 was considered to be significant. Results In vitro cell quality parameters were measured prior to freezing and at 1 and 24 h post-thawing, as shown in Table 1. As expected, there was a significant decrease in platelet number after the freeze/ thaw process (p < 0.0001). However, the average platelet recovery

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Table 1 In vitro quality parameters of cryopreserved platelets.a Pre-freeze 9

Platelet count (10 /L) Platelet recovery (%) MPV (fL) pH (22 °C) Glucose (mmol/L) Lactate (mmol/L) pO2 (mm/Hg) pCO2 (mm/Hg) HCO3 (mmol/L) CD61* microparticles (%) Glycocalicin (AU/mL) HSR (%) Aggregation (% Max) Collagen ADP

Post-thaw 1 h

Post-thaw 24 h

1014 ± 119.9 – 5.1 ± 0.3 7.3 ± 0.1 5.9 ± 0.5 6.6 ± .07 132.8 ± 14.1 16.5 ± 2.8 7.8 ± 0.4 0.8 ± 0.2 466.4 ± 275.0 63.9 ± 18.9

699.3 ± 118.6 68.8 ± 7.1 4.1 ± 0.3* 7.37 ± 0.07* 6.7 ± 3.4 2.7 ± 1.1* 153.2 ± 10.3* 16.5 ± 2.1 10.0 ± 2.7* 4.5 ± 1.5* 834.9 ± 200.7* 14.9 ± 6.6*

773.1 ± 101.3*,# 76.5 ± 5.8 3.8 ± 0.2*,# 7.4 ± 0.1* 5.9 ± 3.3* 3.8 ± 1.1*,# 158.2 ± 9.5* 12.8 ± 1.5*,# 8.7 ± 3.0* 2.57 ± 1.4*,# 1080.4 ± 258.0*,# 24.1 ± 5.5*,#

95.6 ± 9.8 35.5 ± 27.9

41.4 ± 21.1* 6.6 ± 3.8*

30.1 ± 12.6* 10.7 ± 4.4*

75.1 ± 12.4* 21.6 ± 12.9*

81.3 ± 8.4* 14.2 ± 9.6*

Mitochondrial membrane potential Polarised (%) 91.8 ± 2.0 Depolarised (%) 3.5 ± 2.9

*

a

Values shown as mean ± SD; n = 9. Indicates p < 0.05 compared to pre-freeze. # Indicates p < 0.05 compared to 1 h post-thaw MPV, mean platelet volume; HSR, hypotonic shock response; HCO3, bicarbonate. *

was 70% (range 60–80%). The MPV was also decreased at 1 and 24 h post-thawing (p < 0.0001 and p = 0.0003, respectively). The platelet units maintained a pH within the range of 6.4–7.4 (Table 1), as recommended by the Council of Europe and the UK Blood Transfusion Guidelines [12,13]. The partial pressures of oxygen increased, while those of carbon dioxide decreased after thawing (Table 1). The glucose concentration in the platelet concentrates was not significantly different after the freezing and thawing process, but did decrease slightly 24 h post-thawing (Table 1; p < 0.0001), suggesting that platelets were consuming glucose and thus actively metaboliz-

ing. Likewise, the total lactate concentration was significantly reduced immediately after thawing (p < 0.0001), most likely due to the washing steps. However, an increase in the lactate concentration occurred during the 24 h post-thaw period (Table 1). Glucose consumption and lactate production rates were calculated between thawing (1 h) and 24 h storage and were 0.09 mmol/1012 platelets/ h and 0.03 mmol/1012 platelets/h, respectively. In addition, the bicarbonate levels were significantly increased immediately after thawing (p = 0.035), but normalized to pre-freeze values after 24 h (Table 1). Taken together, these results are indicative of active platelet metabolism post-thawing. Platelet morphology and function were affected during the freeze–thaw process, as shown in Table 1. This was evidenced by reduced platelet swirl and a significantly reduced HSR (p = 0.001) immediately after thawing, compared to liquid-stored platelets. However, both of these parameters improved following 24 h incubation (Table 1). There was also an overall decrease in ADP- and collagen-induced aggregation after platelet freeze/thawing (Table 1; p = 0.011 and p = 0.0002, respectively). Further, the percentage of platelet microparticles was significantly higher after platelet thawing (Table 1; p < 0.0001). Calcein-AM and FM4-64 were used to discriminate between viable and non-viable cells. The overall platelet viability was significantly reduced to approximately 50% in the immediate period following thawing (p < 0.0001). However, the percentage of viable cells significantly improved at 24 h post-thawing (Fig. 1A and B; p < 0.0001). During apoptosis, PS is translocated to the platelet surface and a decrease in Dw occurs [33,44]. The percentage of annexin-Vpositive cells was significantly higher than liquid-stored platelets immediately following thawing (Fig. 2A and B; p < 0.0001). Similarly, there was a slight, but significant reduction in Dw after the freeze/thaw process (Table 1; p = 0.005). Interestingly, both apoptotic indicators showed improvement after 24 h.

Fig. 1. Platelet viability is reduced following platelet cryopreservation. Platelets were sampled prior to freezing and at 1 and 24 h post-thaw. The cells were stained with calcein-AM and FM4-64 and analyzed immediately by flow cytometry. (A) The data represent mean ± SD (error bars) of the % of cells positive for calcein-AM (viable) and FM4-64 (non-viable) from nine donors, ⁄Indicates p < 0.05 compared to pre-freeze; #Indicates p < 0.05 compared to 1 h post-thaw. (B) Representative dot plots showing viable (calcein-AMbright/FM4-64dim) and non-viable (calcein-AMdim/FM4-64bright) cell populations.

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Fig. 2. Phosphatidylserine expression is increased following platelet cryopreservation. Platelets were sampled prior to freezing and at 1 and 24 h post-thaw and were stained with annexin-V-FITC and analyzed immediately by flow cytometry. (A) The data represent mean ± SD (error bars) from nine donors of the % of cells positive for annexin-V, ⁄ Indicates p < 0.05 compared to pre-freeze; #Indicates p < 0.05 compared to 1 h post-thaw. (B) Representative histograms demonstrating annexin-V positive staining.

A significant increase in the expression of platelet activation markers, CD62P and CD63, was detected post-thawing (Fig. 3A; p < 0.0001 and p = 0.005, respectively). Further, a decrease in the binding of CD41 and CD42b was observed post-thaw, which is known to occur as a result of platelet activation. Interestingly, two populations of CD42b-positive platelets were present, one displaying normal levels of CD42b and one expressing reduced levels (Fig. 3B). Further, there was a significant increase in glycocalicin in the platelet supernatant, indicating GPIba (CD42b) cleavage (Table

1; p = 0.0004). There was no difference in CD61 and CD47 surface expression between liquid-stored and frozen platelets (data not shown). The secretion of cytokines was measured in the supernatant of platelet concentrates before and after freezing. The data are summarized in Table 2. Briefly, the concentrations of sCD62P, RANTES and EGF were greater in the thawed units, compared to the pre-freeze controls (p < 0.0001, p = 0.001 and p < 0.0001, respectively). In contrast, IL-1b and RANTES were decreased during

Fig. 3. Platelet surface marker expression is altered following cryopreservation. Platelets were sampled prior to freezing and at 1 and 24 h post-thaw and the cells were stained with the indicated antibodies and analyzed by flow cytometry. (A) The data represent mean ± SD (error bars) from nine donors, ⁄Indicates p < 0.05 compared to prefreeze; # Indicates p < 0.05 compared to 1 h post-thaw. (B) Representative histograms demonstrating altered CD42b expression.

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Table 2 Cytokine concentrations in cryopreserved platelets.a Pre-freeze IL-1b (pg/mL) TNF-a (pg/mL) sCD62P (ng/mL) sCD40L (pg/mL) PF4 (ng/mL) PDGF-AB (ng/mL) RANTES (ng/mL) TGF-b (ng/mL) EGF (pg/mL) a * #

25.6 ± 10.7 10.4 ± 24.4 16.6 ± 2.4 1564.1 ± 1288.4 3437.8 ± 427.3 7.2 ± 1.4 35.4 ± 1.6 33.3 ± 6.4 105.8 ± 24.7

Post-thaw 1 h *

13.2 ± 5.5 0.7 ± 1.7 29.1 ± 9.7* 902.1 ± 706.67 3693.0 ± 627.7 7.8 ± 2.4 33.7 ± 1.5* 40.2 ± 12.2 477.7 ± 113.6*

Post-thaw 24 h 11.9 ± 5.9* 0.8 ± 2.3 61.1 ± 9.9*,# 1268.9 ± 770.4* 4893.6 ± 578.7*,# 12.8 ± 1.9*,# 36.1 ± 0.6* 62.1 ± 11.9*,# 719.2 ± 92.8*,#

Values shown as mean ± SD; n = 9. Indicates p < 0.05 compared to pre-freeze. Indicates p < 0.05 compared to 1 h post-thaw.

the immediate post-thaw period. However, by 24 h after thawing, the levels of all platelet-associated cytokines tested had significantly increased. TNF-a levels remained unchanged during the freeze–thaw process. Discussion The results presented here describe a protocol for freezing and thawing buffy-coat-derived platelet concentrates in plasmareduced conditions, by substitution with PAS. Platelet recovery was acceptable, although a deficit of in vitro platelet quality was evident after freezing. The results also suggest that extended incubation post-thawing may alleviate some of these effects. The freezing protocol used in this study was carried out with a combination of plasma and PAS (SSP+). In this study, we found that approximately 50% plasma/SSP+ was the lowest concentration of plasma that could support acceptable platelet recovery. Another study reached a similar conclusion when platelets were frozen in plasma and Composol [9]. Both SSP+ and Composol are composed of similar constituents, although Composol contains gluconate, which acts as a chelator, whilst SSP+ contains phosphate, which increases the buffering capacity of the medium [16,34]. Platelet recovery after freezing was comparable to previously published data [23,28], despite differences in the methods used for freeze/thawing. For standard unfrozen buffy-coat-derived platelet concentrates, the UK guidelines require that 75% of products contain greater than 240  109 platelets per unit [13]. Using this criterion, only 44% of thawed units in this study met this requirement. However, this result is a significant improvement on previously published methods [23]. Alternatively, the Council of Europe guidelines recommend that frozen apheresis platelet concentrates contain greater than 40% of the pre-freeze platelet number [12]. There are not yet any acceptance criteria for the measurement of frozen buffy-coat platelet concentrates. However, applying the Council of Europe acceptance criteria used for apheresis platelets, 100% of the frozen platelet concentrates produced in this study would be considered acceptable. Multiple changes to platelet morphology were detected postthawing. For example, the platelets shrank slightly during the process, as indicated by a reduction in the MPV. Further, the post-thaw loss of swirl suggests that a large proportion of platelets had changed from a discoid to a spherical shape [5]. However, extended incubation at 22 °C with agitation enabled recovery of the morphological defects. The frozen platelets also had an extremely low mean HSR value of between 15% and 24% after thawing, compared with the liquid-stored platelets prior to freezing. However, these results are comparable to reports that examined frozen platelets reconstituted in 100% plasma [23,40], suggesting that reducing the plasma levels to 50% does not adversely affect platelet quality. Metabolic and pH disruptions in stored platelets lead to an inability to maintain normal shape and response to hypotonic

shock [21]. Previous studies have focused on pH levels post-thaw, but have not assessed metabolic parameters such as pO2, pCO2, glucose, lactate and bicarbonate concentrations. Maintaining high pO2 and low pCO2 is crucial during platelet storage because hypoxia may lead to increased glycolysis, lactate production, and subsequent pH decrease [47]. The pH was maintained well above the Council of Europe recommendations (pH P6.4) in all units [12], and was similar to previous studies using plasma to reconstitute thawed platelets [23,28]. The glucose consumption was elevated approximately twofold compared to the limits deemed acceptable by other groups for in vitro platelet storage [11,15,29]. The higher level of glucose consumption was most likely to have been due to platelet stress during the thawing process, as the published rates are for liquid-stored platelets. However the lactate production is within acceptable limits, and may be due to the presence of citrate in the PAS [11,14]. The data presented here shows that frozen platelets were capable of active metabolism after thawing and storage for 24 h. Platelets are known to show a rapid reduction in their responsiveness to aggregating agents during storage ex vivo [31] and following cryopreservation [4,23,24,32,38,40]. These data are consistent with the findings reported here. However, although dampened, the ability to aggregate was not completely abolished by the freeze/thaw process. Further, it has been suggested that reduced in vitro ADP-induced aggregation is reversed after transfusion [31]. Platelet activation in vitro may reduce aggregation [29] as well as the survival of transfused platelets. As shown in this and previous studies, P-selectin (CD62P) expression is markedly up-regulated following cryopreservation [3,4,9,32]. A threshold value of 40% CD62P-positive platelets has previously been used as the limit of acceptability [3], as higher CD62P levels have been associated with clearance of platelets by the reticuloendothelial system [22]. Using this criterion, the frozen platelets in this study were acceptable. In addition, the frozen platelets also displayed increased CD63 expression, decreased GPIIb (CD41a), and GPIba (CD42b) expression. This study and others have shown that cryopreservation results in a sub-population of platelets expressing low levels of GPIba [4,9,32]. This may have in vivo implications as post-transfusion platelet clearance is thought to be mediated by GPIba, where hepatic macrophages recognize and remove platelets expressing altered GPIba [35]. However, more than 50% of platelets retained GPIba expression, suggesting that platelet adhesion under flow should not be affected [43]. Consistent with the reduction in platelet viability postcryopreservation, there was also a significant increase in PS externalization [10,30,39]. Exposure of PS on the outer leaflet of the cell membrane plays an important role in the recognition and subsequent clearance of senescent platelets [7]. Alteration of the Dw was also used to assess platelet apoptosis. Although the freeze/ thaw process did lead to depolarization of the Dw, the percentage of cells was quite low (approximately 20%) and improved during the 24 h storage period. The changes in annexin-V binding mirrored the Dw, with an initial peak immediately after thawing, followed by a degree of recovery. Platelet activation and apoptosis also lead to release of cytokines from the storage granules of platelets. A comprehensive analysis of the secretion of granule contents has not previously been undertaken post freeze/thawing. These results demonstrate an increase in the levels of sCD62P, RANTES and EGF immediately after thawing and a significant increase in the levels of all plateletassociated cytokines by 24 h post-thaw. The clinical impact of infusing cytokines into patients when transfusing platelet concentrates is not yet fully understood [45], however, it is known that RANTES and TGF-b1 exert profound effects on the immune system [20] and high levels of RANTES have been involved in severe

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allergic transfusion reactions [46]. Further, high concentrations of sCD40L have also been associated with adverse transfusion reactions [6]. Importantly, there were no observed increases in the level of this proinflammatory cytokine following freezing. Currently, frozen platelet units must be transfused within 2–6 h after thawing or reconstitution [9,23,24]. This is primarily due to the open-system required during the processing procedure, which may result in bacterial contamination. Our study and one previous study have examined platelet quality after 24 h [9]. These results demonstrate that although some quality parameters continue to deteriorate during the 24 h storage period, others, such as swirl, platelet count, PS expression and normalization of GPIba expression actually improved. Therefore, if a closed system could be created, perhaps by the production of an off-the-shelf cryoprotectant based on an optimal PAS, extension of the transfusion window for these products may be realistic. To summarize, these results demonstrate that platelets frozen in Me2SO and reconstituted in SSP+ do have a deficit of their in vitro quality parameters, when compared to liquid-stored units. However, these results are comparable to, if not better than, previously published data, suggesting that reducing the plasma level is not detrimental. Further, cryopreserved platelets, frozen using less sophisticated methods, have been used clinically for more than 30 years [36] and it has been shown that frozen platelets are more effective than liquid-preserved platelets in restoring hemostasis and reducing nonsurgical blood loss [24,28]. Disclosure statement The authors have no conflict of interest to disclose.

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