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Platelet additive solutions: A future perspective
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Transfusion Clinique et Biologique 14 (2007) 522–525 http://france.elsevier.com/direct/TRACLI/
Original article
Platelet additive solutions: A future perspective Solutions additives de conservation des plaquettes : perspectives futures P.F. van der Meer Sanquin Blood Bank North West region, Plesmanlaan 125, 1006 CX Amsterdam, PO Box 9137, 1006 AC Amsterdam, The Netherlands Available online 16 April 2008
Abstract Platelet additive solutions (PASs) were first developed in the 1980s, and continued to be improved over the following years. The use of PASs as replacement for plasma has a number of benefits, both for the quality of the platelet concentrates and for the patients. However, some PASs have been associated with a lower platelet yield in the PCs, a shorter storage time, and a lower increment in the patient when compared to PCs in plasma. A number of reformulations of the PASs have taken place to counteract these disadvantages. Most PASs use acetate as nutrient for the platelets, which has the benefit of generating bicarbonate when oxidized by the platelets, thus supplying its own buffering capacity. Alternatively, glucose is used, but may cause deterioration of pH in the stored PCs due to the formation of lactic acid. Addition of other buffering substances, such as phosphate, can be added to ensure maintenance of neutral pH. An important finding was the inhibiting effect of potassium and magnesium on platelet activation. The initially developed PASs lacked these two ingredients and showed reduced storage times of the PCs in PAS when compared to those stored in plasma. However, when these constituents are included in the PAS, storage time is similar and even exceeds those seen for PCs in plasma. Considerable research is done in further formulating the optimal PAS. Bicarbonate is being considered as buffer for these PASs. Also, L– carnitine appears to have a favorable effect on stored platelets, including a reduction of platelet metabolism, and inhibition of apoptosis. Another area of optimization is lowering of plasma content needed for maintaining platelet quality. Where current PASs still need at least 30% residual plasma, there is a trend towards lowering the plasma content to less than 5% with the newer PASs. Preservation of purinergic platelet receptor functionality by ADP-degrading activities in plasma appears to play an important role in this respect. Development of PASs are usually based on in vitro studies alone. It is important to realize that only clinical studies can give definitive answers about the quality of platelets stored in PASs. Sofar, only limited clinical evaluations have been published that either studied the effectiveness of platelets in initially-developed PASs, or were specifically done in combination with pathogen reduction technologies. Thus, PASs seem to be an excellent replacement for (part of) the plasma when producing PCs, and allow extended storage with maintenance of quality, but more clinical studies are needed to substantiate in vitro results. # 2008 Elsevier Masson SAS. All rights reserved. Résumé Les solutions additives de conservation des plaquettes (SAP) ont été introduites dans les années 1980 et s’améliorent encore au fil des ans. L’utilisation de SAP en remplacement d’une partie du plasma présente de nombreux avantages, à la fois pour la qualité des concentrés plaquettaires et pour le malade. Cependant, certaines SAP entraînent une diminution du nombre de plaquettes dans des concentrés plaquettaires (CP), une diminution de la durée de vie et une augmentation plus faible du taux plaquettaire chez les maladies par comparaison aux CP en plasma. De nombreuses reformulations des SAP ont été proposées pour contrecarrer ces désavantages. La plupart des SAP contiennent de l’acétate comme nutriment plaquettaire, ce qui permet après oxydation de générer du bicarbonate, essentiel dans le maintien du pouvoir tampon. Le glucose peut également être utilisé, mais il entraîne une détérioration du pH dans les CP stockés à cause de la formation d’acide lactique. L’addition d’autres substances tampons, comme le phosphate, peut assurer le maintien d’un pH neutre. La découverte de l’effet inhibiteur du potassium et du magnésium sur l’activation plaquettaire a été une étape importante. Les premiers SAP étaient dépourvus de ces ingrédients et leur temps de conservation étaient limités par rapport aux CP en plasma. Cependant, après addition de ces substances dans les SAP, le temps de conservation des plaquettes devient égal et même supérieur à celui des plaquettes en plasma. De nombreuses recherches sont entreprises pour formuler une SAP optimale. Le bicarbonate est le tampon choisi pour ces SAP. De même, la L–carnitine possède un effet bénéfique sur la conservation des plaquettes, en incluant une réduction du métabolisme plaquettaire et une inhibition de l’apoptose. Une autre voie d’optimisation est l’abaissement de la quantité de plasma nécessaire au maintien de la qualité plaquettaire. Bien que les SAP classiques nécessitent 30 % de plasma résiduel, la tendance actuelle dans les nouveaux SAP est une reduction à 5 %. La conservation d’un récepteur plaquettaire putinergique fonctionnel par dégradation de E-mail address: [email protected]. 1246-7820/$ – see front matter # 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.tracli.2008.03.004
P.F. van der Meer / Transfusion Clinique et Biologique 14 (2007) 522–525
523
l’ADP dans le plasma semble jouer un rôle important à cet égard. Le développement de SAP s’effectue généralement par des études in vitro. Cependant, seules des études cliniques peuvent fournir des réponses définitives sur la qualité des plaquettes conservées en SAP. Actuellement, il n’existe qu’un nombre limité d’évaluations cliniques publiées qui concernent soit l’efficacité des plaquettes conservées dans les premières SAP décrites, soit des études en relation avec les technologies de réduction des pathogènes. En définitive, les SAP semblent être excellentes pour le remplacement (partiel) du plasma des CP ; elles permettent une augmentation du temps de conservation avec maintien de la qualité, mais plus d’études cliniques seraient nécessaires pour étayer les études in vitro. # 2008 Elsevier Masson SAS. All rights reserved. Keywords: Platelet storage; Additive solutions; Transfusion Mots clés : Solutions additives ; Conservation des plaquettes ; Transfusion
1. Benefits and disadvantages of platelet additive solutions The development of platelet additive solutions (PASs) was initiated in the 1980s [1,2], and further modifications and improvements followed in subsequent years. For the production of platelet concentrates (PCs), the replacement of plasma with a PAS has a number of potential benefits. First of all, PASs can be produced sterile and pathogen-free, and have a standardized composition, contrasting to plasma that has inherent donor variance. Furthermore, though current PASs still require at least 30% residual plasma in the PC to maintain platelet quality, the protein content is much lower, thereby reducing allergic reactions. Also, due to absence of antibodies, AB0-incompatible transfusions can be tolerated easier, and the risk of TRALI is reduced. By not using plasma for the preparation of PCs, more becomes available for fractionation. Finally, specific PASs have been developed to allow pathogen reduction technologies, both because plasma may inhibit the effectiveness of the pathogen reduction treatment, and to maintain platelet quality after treatment. Initially, however, there were disadvantages associated with the use of PAS. In the buffy coat method, the centrifugation of the buffy coat pool is more difficult due to the lower viscosity of PAS, and therefore the platelet yield is generally 10–15% lower as when plasma is used. As will be discussed below in more detail, some PASs exhibit a shorter storage time, as well as show a lower increment in the patient when compared to PCs in plasma. 2. Acetate and glucose as platelet nutrient Most PCs are still made with CPD-anticoagulated plasma, although more and more countries consider the use of PAS [3]. When compared to ‘‘normal’’ plasma, the phosphate content of CDP-plasma is about four times higher, and glucose content is five to six-fold higher [1]. When platelets are stored in CPDplasma, glucose serves as the main nutrient. When PAS is used, a substitute platelet nutrient has to be included. Although initially glucose was used in PAS [1] these solutions are difficult to sterilize at neutral pH due to caramellization of glucose. Also, when oxidized, glucose will generate lactic acid, causing rapid deterioration of pH in the stored PCs. Hence, both in CPD plasma and in PASs, the presence of high amounts of glucose should be avoided, and most PASs therefore use acetate as platelet nutrient [4]. Acetate has the added benefit that it
generates bicarbonate when oxidized by the platelets, thus supplying its own buffering capacity [5]. One widely-used PAS containing acetate is PASII (T-Sol, Baxter, Nivelles, Belgium). The composition of this solution, as well as other PASs discussed in this paper, are summarized in Table 1. Gulliksson et al. [5] showed that PCs in PASII could be stored satisfactorily for at least five days. In fact, still on Day 9, pH was 6.96 0.06, while other biochemical parameters showed acceptable values. In the following years, however, doubt was cast on the clinical effectiveness of these platelets. Already in 1996, Turner et al. [6] showed that platelet survival and recovery in volunteer donors were reduced compared to plasma. After five days of storage of the PCs, platelet survival in plasma was 6.5 1.5 days, versus 5.1 1.3 days in PASII ( p < 0.01). Platelet recovery was 51.1 15.9% for platelets stored in plasma, which was significantly better than found in PASII, 29.8 13.5% ( p < 0.01). In 2000, De Wildt-Eggen et al. [7] showed in a randomized study comparing platelets stored in plasma with those stored in PASII, that those in PASII had lower corrected count increments (CCI). For PCs that had been stored for four to five days, the 1 h CCI was 19.9 6.7 for platelets in plasma (n = 97) versus 16.5 6.7 in PASII (n = 62; p < 0.05). The 20-h CCI was not different: 10.0 6.9 for plasma and 9.1 6.7 for PASII. The percentage transfusions associated with an allergic reaction decreased from 12% for platelets in plasma to 5.3% in PASII ( p < 0.05). These lower increments were recently confirmed by Kerkhoffs et al. [8] who found an average 1 h CCI of 13.9 7.0 for platelets in plasma (n = 311) versus 11.2 6.4 for those in PASII (n = 373; p = 0.004). Again, the 24 h CCIs Table 1 Composition of various platelet additive solutions (in mmol/L) Commercial name Alternative name
TSol PASII
Composol GAC
InterSol PASIII
SSP+ PASIIIM
GASP-BIC
NaCl Acetate KCl MgCl2 Na2HPO4 Na3-citrate Citric acid Gluconate Glucose pH
115.5 30 – – – 10 – – – 7.2
90 27 5 3 – 10 – 30 – 7.0
77.3 28.2 – – 28.2 10.8 – – – 7.2
69 41 5 1.5 26 10 – – – 7.2
110 15 5 3 4 – 7.5 – 30 5.2
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P.F. van der Meer / Transfusion Clinique et Biologique 14 (2007) 522–525
were not significantly different: 8.4 6.9 for plasma and 6.8 6.4 for PASII. The percentage allergic reactions decreased from 5.5% in plasma-PCs to 2.4% PASII-PCs ( p = 0.04). Multivariate analysis of transfusion failures (i.e. a 1 h CCI < 7.5 or a 24 h CCI < 4.5) showed a significant effect of infection, enlarged spleen and use of ATG on the 1 h CCI, and of fever, enlarged spleen, ATG and the recipient’s age on the 24 h CCI. Storage time and storage medium had no significant effect on the 1 and 24 h CCI. So, although the CCIs of platelets stored in PASII are lower than those in plasma, the clinical relevance of this finding is probably not very large. 3. Inhibition of platelet activation by potassium and magnesium De Wildt-Eggen et al. [9] elegantly demonstrated the beneficial effect of potassium and magnesium –and the two combined– on platelet activation. Using PASII as basis for platelet storage, the addition of 4.5 mmol/L potassium resulted in a pH on Day 7 of 7.13 0.03, versus 6.93 0.04 for PASII alone ( p < 0.001). When 1.5 mmol/L magnesium was added, Day 7 pH was 7.10 0.07 versus 6.98 0.07 for PASII alone ( p < 0.05). Finally, the two combined gave a pH of 7.15 0.10 on Day 7 of storage of the PCs in PASII with potassium and magnesium, versus 6.94 0.05 in PASII alone ( p < 0.05). A notable difference was found in the platelet activation, measured as CD62P expression, that was 23 6% for platelets in PASII with potassium and magnesium versus 50 8% for platelets stored in PASII alone ( p < 0.001). As reference, platelets stored for seven days in plasma had a pH of 7.03 0.06 ( p < 0.05 versus PASII with potassium and magnesium) and a CD62P expression of 35 8% ( p < 0.05). For a similar PAS with potassium and magnesium, Composol, a storage time of at least 12 days could be demonstrated with maintenance of in vitro quality [10]. So, addition of potassium and magnesium inhibits CD62P expression [11], and allows extended platelet storage beyond the currently accepted five or seven days storage intervals. 4. Alternative buffers Though bicarbonate is an excellent buffer, the amount available to prevent acidification of the storage medium may not be sufficient under all conditions. Specifically for pathogen reduction, InterSol (also known as PASIII, Baxter) has been developed [12]. This PAS contains phosphate to maintain neutral pH after the pathogen reduction procedure. However, as demonstrated by Gulliksson et al. [13], phosphate stimulates platelet metabolism. Comparing PASII with PASIII, glucose consumption was 0.06 0.02 mmol/1011 platelets/day in PASII versus 0.08 0.02 mmol/1011 platelets/day for PASIII ( p < 0.001). Lactate production showed the same effect: 0.11 0.03 versus 0.14 0.04 mmol/1011 platelets/day, respectively ( p < 0.001), for PASII and PASIII. Despite this effect, pH remained almost unaffected, 6.97 0.15 in PASII versus 6.94 0.07 for PASIII (not significant), due to the strong buffering capacity of phosphate.
A PAS containing phosphate as well as potassium and magnesium showed possibilities for storage up to 16 days. In a pilot study evaluating storage of platelets in SSP+ (MacoPharma, Toircoing, France), pH was still 7.19 0.06 on Day 16 [14]. Other in vitro markers also indicated good in vitro quality on Day 16, with a CD62P expression of 26 6%, PS exposure of 24 4% and a hypotonic shock-response of 50 15%. In recent studies, bicarbonate is being reconsidered as buffer. Sweeney et al. [15] used bicarbonate pills in a platelet storage container in combination with a glucose-containing PAS with a pH of about 5.2, GASP-BIC. Using PRP-derived platelet concentrates, pH was neutral at 7.07 0.15 on the day of production, compared to 7.45 0.04 for platelets stored in plasma ( p < 0.01). At Day 7 of storage, pH was well maintained at 7.27 0.24 for the bicarbonate-containing PAS, versus 7.19 0.09 for plasma (not significant). Further, CD62P expression was 6 4% for platelets in this PAS, versus 18 10% in plasma ( p < 0.01). Bicarbonate was still present on Day 7:8 1 mmol/L for the PAS and 8 2 mmol/L for the PCs in plasma (not significant). 5. Further developments Instead of using PAS simply as a suspension medium for platelets, nowadays PAS can be considered as a ‘designer solution’ where ingredients can be added to specifically influence certain characteristics of platelet storage. A possible candidate for further development in this respect is L–carnitine. Sweeney et al. showed [16], for platelet concentrates in plasma, that the addition of 5 mmol/L L–carnitine considerably affected storage characteristics. At day 10 of storage, pH was 6.64 0.20 for PCs with L–carnitine, versus 6.42 0.30 for those without ( p < 0.01). This is probably due to inhibition of platelet metabolism by L–carnitine [17], giving lower lactic acid formation. Because L–carnitine has been associated with inhibition of apoptosis by regulating the activity of caspases [18], further studies are warranted. As early as 1996, Klinger et al. [19] demonstrated that at least 30% plasma was required to maintain in vitro quality for platelets stored in PAS. A study by the BEST Collaborative [20] showed a deleterious effect on in vitro measures already if the residual plasma content was lowered from 30 to 20%. Recently, Cauwenberghs et al. [21] showed that washing platelets with a plasma-free buffer rapidly resulted in unresponsiveness to ADP stimulation. She elucidated that ATP- and ADP-degrading activities in plasma preserved the purinergic receptor activities of stored platelets. She was able to show that a substantial part of the ATP-degrading activity in plasma could be inhibited by treatment with anti-CD39 antibody. Although application of an antibody in a PAS is probably difficult to realize, possibly other ways can be found to establish the same effect. If a feasible solution can been found, the residual plasma content may be lowered to less than 5%. Such low percentages residual plasma would decrease adverse effects in patients, notably allergic reactions. In addition, the risk of TRALI would decrease, and AB0-incompatible platelet transfusions could be given easier as no AB0-antibodies are present in the storage medium.
P.F. van der Meer / Transfusion Clinique et Biologique 14 (2007) 522–525
6. Clinical evaluation of PAS Many studies with PAS only report in vitro data. Although these studies are valuable in the development of new PASs, a follow-up with clinical studies is necessary. So far, only limited clinical evaluations have been published that studied the effectiveness of platelets stored in PASs that were developed in the late 1990s. More recently-developed PASs were specifically investigated only in combination with pathogen reduction technologies. Many of these clinical studies targeted at pathogen-reduction used ‘‘routinelyproduced’’ platelet concentrates as control groups. These, however, are not necessarily the best control group. More often than not, these control groups consisted of platelets stored in PASII, which are known to give lower CCIs [6–8]. As Murphy pointed out [22], this may result in a ‘‘creeping inferiority’’ in the quality of PCs. Therefore, transfusions with fresh PCs in plasma should be included in some form in clinical evaluations of new platelet products in additive solution, to allow fair comparison and to prevent this downward creep. In summary, PASs seem to be an excellent replacement for part of the plasma when producing PCs. Extended storage with maintenance of in vitro quality seems to be feasible. Also, further reduction of the plasma content to below 5% seems to be within reach. Further in vitro studies with respect to the composition of PASs are warranted, and PCs produced with and stored in these newly developed PASs should be evaluated in clinical studies with the right control groups to substantiate in vitro results. References [1] Holme S, Heaton WA, Courtright M. Improved in vivo and in vitro viability of platelet concentrates stored for seven days in a platelet additive solution. Br J Haematol 1987;66:233–8. [2] Rock G, White J, Labow R. Storage of platelets in balanced salt solutions: a simple platelet storage medium. Transfusion 1991;31:21–5. [3] Murphy S. Platelets from pooled buffy coats: an update. Transfusion 2005;45:634–9. [4] Gulliksson H, Eriksson L, Högman CF, Payrat JM. Buffy-coat-derived platelet concentrates prepared from half-strength citrate CPD and CPD whole-blood units. Comparison between three additive solutions: in vitro studies. Vox Sang 1995;68:152–9. [5] Bertolini F, Murphy S, Rebulla P, Sirchia G. Role of acetate during platelet storage in a synthetic medium. Transfusion 1992;32:152–6.
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[6] Turner VS, Mitchell SG, Hawker RJ. More on the comparison of PlasmaLyte A and PAS 2 as platelet additive solutions. Transfusion 1996;36: 1033–4 (Letter). [7] De Wildt-Eggen J, Nauta S, Schrijver JG, van Marwijk Kooy M, Bins M, van Prooijen HC. Reactions and platelet increments after transfusion of platelet concentrates in plasma or an additive solution: a prospective, randomized study. Transfusion 2000;40:398–403. [8] Kerkhoffs JL, Eikenboom JC, Schipperus MS, van Wordragen-Vlaswinkel RJ, Brand R, Harvey MS, et al. A multicenter randomized study of the efficacy of transfusions with platelets stored in platelet additive solution II versus plasma. Blood 2006;108:3210–5. [9] De Wildt-Eggen J, Schrijver JG, Bins M, Gulliksson H. Storage of platelets in additive solutions: effects of magnesium and/or potassium. Transfusion 2002;42:76–80. [10] Van der Meer PF, Pietersz RN, Reesink HW. Storage of platelets in additive solution for up to 12 days with maintenance of good in-vitro quality. Transfusion 2004;44:1204–11. [11] Gawaz M, Ott I, Reininger AJ, Neumann FJ. Effects of magnesium on platelet aggregation and adhesion. Magnesium modulates surface expression of glycoproteins on platelets in vitro and ex vivo. Thromb Haemost 1994;72:912–8. [12] Lin L, Dikeman R, Molini B, Lukehart SA, Lane R, Dupuis K, et al. Photochemical treatment of platelet concentrates with amotosalen and long-wavelength ultraviolet light inactivates a broad spectrum of pathogenic bacteria. Transfusion 2004;44:1496–504. [13] Gulliksson H, Larsson S, Kumlien G, Shanwell A. Storage of platelets in additive solutions: effects of phosphate. Vox Sang 2000;78:176–84. [14] Van der meer PF, Eijzenga-Demmendal M, Pietersz RN. Platelets stored at 4 8C, an evaluation of platelets in SSP+ additive solution. Transfusion 2007;47(Suppl.):84A (Abstract). [15] Sweeney J, Kouttab N, Holme S, Kurtis J, Cheves T, Nelson E. Storage of platelet-rich plasma-derived platelet concentrate pools in plasma and additive solution. Transfusion 2006;46:835–40. [16] Sweeney JD, Blair AJ, Cheves TA, Dottori S, Arduini A. L–carnitine decreases glycolysis in liquid-stored platelets. Transfusion 2000;40:1313–9. [17] Sweeney JD, Arduini A. L–carnitine and its possible role in red cell and platelet storage. Transfus Med Rev 2004;18:58–65. [18] Mutomba MC, Yuan H, Konyavko M, Adachi S, Yokoyama CB, Esser V, et al. Regulation of the activity of caspases by L–carnitine and palmitoylcarnitine. FEBS Lett 2000;478:19–25. [19] Klinger MH, Josch M, Klüter H. Platelets stored in a glucose-free additive solution or in autologous plasma–an ultrastructural and morphometric evaluation. Vox Sang 1996;71:13–20. [20] Gulliksson H, AuBuchon JP, Cardigan R, van der Meer PF, Murphy S, Prowse C, et al. Storage of platelets in additive solutions: a multicentre study of the in vitro effects of potassium and magnesium. Vox Sang 2003;85:199–205. [21] Cauwenberghs S, Feijge MA, Hageman G, Hoylaerts M, Akkerman JW, Curvers J, et al. Plasma ectonucleotidases prevent desensitization of purinergic receptors in stored platelets: importance for platelet activity during thrombus formation. Transfusion 2006;46:1018–28. [22] Murphy S. Radiolabeling of PLTs to assess viability: a proposal for a standard. Transfusion 2004;44:131–3.
Transfusion Clinique et Biologique 14 (2007) 522–525 http://france.elsevier.com/direct/TRACLI/
Original article
Platelet additive solutions: A future perspective Solutions additives de conservation des plaquettes : perspectives futures P.F. van der Meer Sanquin Blood Bank North West region, Plesmanlaan 125, 1006 CX Amsterdam, PO Box 9137, 1006 AC Amsterdam, The Netherlands Available online 16 April 2008
Abstract Platelet additive solutions (PASs) were first developed in the 1980s, and continued to be improved over the following years. The use of PASs as replacement for plasma has a number of benefits, both for the quality of the platelet concentrates and for the patients. However, some PASs have been associated with a lower platelet yield in the PCs, a shorter storage time, and a lower increment in the patient when compared to PCs in plasma. A number of reformulations of the PASs have taken place to counteract these disadvantages. Most PASs use acetate as nutrient for the platelets, which has the benefit of generating bicarbonate when oxidized by the platelets, thus supplying its own buffering capacity. Alternatively, glucose is used, but may cause deterioration of pH in the stored PCs due to the formation of lactic acid. Addition of other buffering substances, such as phosphate, can be added to ensure maintenance of neutral pH. An important finding was the inhibiting effect of potassium and magnesium on platelet activation. The initially developed PASs lacked these two ingredients and showed reduced storage times of the PCs in PAS when compared to those stored in plasma. However, when these constituents are included in the PAS, storage time is similar and even exceeds those seen for PCs in plasma. Considerable research is done in further formulating the optimal PAS. Bicarbonate is being considered as buffer for these PASs. Also, L– carnitine appears to have a favorable effect on stored platelets, including a reduction of platelet metabolism, and inhibition of apoptosis. Another area of optimization is lowering of plasma content needed for maintaining platelet quality. Where current PASs still need at least 30% residual plasma, there is a trend towards lowering the plasma content to less than 5% with the newer PASs. Preservation of purinergic platelet receptor functionality by ADP-degrading activities in plasma appears to play an important role in this respect. Development of PASs are usually based on in vitro studies alone. It is important to realize that only clinical studies can give definitive answers about the quality of platelets stored in PASs. Sofar, only limited clinical evaluations have been published that either studied the effectiveness of platelets in initially-developed PASs, or were specifically done in combination with pathogen reduction technologies. Thus, PASs seem to be an excellent replacement for (part of) the plasma when producing PCs, and allow extended storage with maintenance of quality, but more clinical studies are needed to substantiate in vitro results. # 2008 Elsevier Masson SAS. All rights reserved. Résumé Les solutions additives de conservation des plaquettes (SAP) ont été introduites dans les années 1980 et s’améliorent encore au fil des ans. L’utilisation de SAP en remplacement d’une partie du plasma présente de nombreux avantages, à la fois pour la qualité des concentrés plaquettaires et pour le malade. Cependant, certaines SAP entraînent une diminution du nombre de plaquettes dans des concentrés plaquettaires (CP), une diminution de la durée de vie et une augmentation plus faible du taux plaquettaire chez les maladies par comparaison aux CP en plasma. De nombreuses reformulations des SAP ont été proposées pour contrecarrer ces désavantages. La plupart des SAP contiennent de l’acétate comme nutriment plaquettaire, ce qui permet après oxydation de générer du bicarbonate, essentiel dans le maintien du pouvoir tampon. Le glucose peut également être utilisé, mais il entraîne une détérioration du pH dans les CP stockés à cause de la formation d’acide lactique. L’addition d’autres substances tampons, comme le phosphate, peut assurer le maintien d’un pH neutre. La découverte de l’effet inhibiteur du potassium et du magnésium sur l’activation plaquettaire a été une étape importante. Les premiers SAP étaient dépourvus de ces ingrédients et leur temps de conservation étaient limités par rapport aux CP en plasma. Cependant, après addition de ces substances dans les SAP, le temps de conservation des plaquettes devient égal et même supérieur à celui des plaquettes en plasma. De nombreuses recherches sont entreprises pour formuler une SAP optimale. Le bicarbonate est le tampon choisi pour ces SAP. De même, la L–carnitine possède un effet bénéfique sur la conservation des plaquettes, en incluant une réduction du métabolisme plaquettaire et une inhibition de l’apoptose. Une autre voie d’optimisation est l’abaissement de la quantité de plasma nécessaire au maintien de la qualité plaquettaire. Bien que les SAP classiques nécessitent 30 % de plasma résiduel, la tendance actuelle dans les nouveaux SAP est une reduction à 5 %. La conservation d’un récepteur plaquettaire putinergique fonctionnel par dégradation de E-mail address: [email protected]. 1246-7820/$ – see front matter # 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.tracli.2008.03.004
P.F. van der Meer / Transfusion Clinique et Biologique 14 (2007) 522–525
523
l’ADP dans le plasma semble jouer un rôle important à cet égard. Le développement de SAP s’effectue généralement par des études in vitro. Cependant, seules des études cliniques peuvent fournir des réponses définitives sur la qualité des plaquettes conservées en SAP. Actuellement, il n’existe qu’un nombre limité d’évaluations cliniques publiées qui concernent soit l’efficacité des plaquettes conservées dans les premières SAP décrites, soit des études en relation avec les technologies de réduction des pathogènes. En définitive, les SAP semblent être excellentes pour le remplacement (partiel) du plasma des CP ; elles permettent une augmentation du temps de conservation avec maintien de la qualité, mais plus d’études cliniques seraient nécessaires pour étayer les études in vitro. # 2008 Elsevier Masson SAS. All rights reserved. Keywords: Platelet storage; Additive solutions; Transfusion Mots clés : Solutions additives ; Conservation des plaquettes ; Transfusion
1. Benefits and disadvantages of platelet additive solutions The development of platelet additive solutions (PASs) was initiated in the 1980s [1,2], and further modifications and improvements followed in subsequent years. For the production of platelet concentrates (PCs), the replacement of plasma with a PAS has a number of potential benefits. First of all, PASs can be produced sterile and pathogen-free, and have a standardized composition, contrasting to plasma that has inherent donor variance. Furthermore, though current PASs still require at least 30% residual plasma in the PC to maintain platelet quality, the protein content is much lower, thereby reducing allergic reactions. Also, due to absence of antibodies, AB0-incompatible transfusions can be tolerated easier, and the risk of TRALI is reduced. By not using plasma for the preparation of PCs, more becomes available for fractionation. Finally, specific PASs have been developed to allow pathogen reduction technologies, both because plasma may inhibit the effectiveness of the pathogen reduction treatment, and to maintain platelet quality after treatment. Initially, however, there were disadvantages associated with the use of PAS. In the buffy coat method, the centrifugation of the buffy coat pool is more difficult due to the lower viscosity of PAS, and therefore the platelet yield is generally 10–15% lower as when plasma is used. As will be discussed below in more detail, some PASs exhibit a shorter storage time, as well as show a lower increment in the patient when compared to PCs in plasma. 2. Acetate and glucose as platelet nutrient Most PCs are still made with CPD-anticoagulated plasma, although more and more countries consider the use of PAS [3]. When compared to ‘‘normal’’ plasma, the phosphate content of CDP-plasma is about four times higher, and glucose content is five to six-fold higher [1]. When platelets are stored in CPDplasma, glucose serves as the main nutrient. When PAS is used, a substitute platelet nutrient has to be included. Although initially glucose was used in PAS [1] these solutions are difficult to sterilize at neutral pH due to caramellization of glucose. Also, when oxidized, glucose will generate lactic acid, causing rapid deterioration of pH in the stored PCs. Hence, both in CPD plasma and in PASs, the presence of high amounts of glucose should be avoided, and most PASs therefore use acetate as platelet nutrient [4]. Acetate has the added benefit that it
generates bicarbonate when oxidized by the platelets, thus supplying its own buffering capacity [5]. One widely-used PAS containing acetate is PASII (T-Sol, Baxter, Nivelles, Belgium). The composition of this solution, as well as other PASs discussed in this paper, are summarized in Table 1. Gulliksson et al. [5] showed that PCs in PASII could be stored satisfactorily for at least five days. In fact, still on Day 9, pH was 6.96 0.06, while other biochemical parameters showed acceptable values. In the following years, however, doubt was cast on the clinical effectiveness of these platelets. Already in 1996, Turner et al. [6] showed that platelet survival and recovery in volunteer donors were reduced compared to plasma. After five days of storage of the PCs, platelet survival in plasma was 6.5 1.5 days, versus 5.1 1.3 days in PASII ( p < 0.01). Platelet recovery was 51.1 15.9% for platelets stored in plasma, which was significantly better than found in PASII, 29.8 13.5% ( p < 0.01). In 2000, De Wildt-Eggen et al. [7] showed in a randomized study comparing platelets stored in plasma with those stored in PASII, that those in PASII had lower corrected count increments (CCI). For PCs that had been stored for four to five days, the 1 h CCI was 19.9 6.7 for platelets in plasma (n = 97) versus 16.5 6.7 in PASII (n = 62; p < 0.05). The 20-h CCI was not different: 10.0 6.9 for plasma and 9.1 6.7 for PASII. The percentage transfusions associated with an allergic reaction decreased from 12% for platelets in plasma to 5.3% in PASII ( p < 0.05). These lower increments were recently confirmed by Kerkhoffs et al. [8] who found an average 1 h CCI of 13.9 7.0 for platelets in plasma (n = 311) versus 11.2 6.4 for those in PASII (n = 373; p = 0.004). Again, the 24 h CCIs Table 1 Composition of various platelet additive solutions (in mmol/L) Commercial name Alternative name
TSol PASII
Composol GAC
InterSol PASIII
SSP+ PASIIIM
GASP-BIC
NaCl Acetate KCl MgCl2 Na2HPO4 Na3-citrate Citric acid Gluconate Glucose pH
115.5 30 – – – 10 – – – 7.2
90 27 5 3 – 10 – 30 – 7.0
77.3 28.2 – – 28.2 10.8 – – – 7.2
69 41 5 1.5 26 10 – – – 7.2
110 15 5 3 4 – 7.5 – 30 5.2
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were not significantly different: 8.4 6.9 for plasma and 6.8 6.4 for PASII. The percentage allergic reactions decreased from 5.5% in plasma-PCs to 2.4% PASII-PCs ( p = 0.04). Multivariate analysis of transfusion failures (i.e. a 1 h CCI < 7.5 or a 24 h CCI < 4.5) showed a significant effect of infection, enlarged spleen and use of ATG on the 1 h CCI, and of fever, enlarged spleen, ATG and the recipient’s age on the 24 h CCI. Storage time and storage medium had no significant effect on the 1 and 24 h CCI. So, although the CCIs of platelets stored in PASII are lower than those in plasma, the clinical relevance of this finding is probably not very large. 3. Inhibition of platelet activation by potassium and magnesium De Wildt-Eggen et al. [9] elegantly demonstrated the beneficial effect of potassium and magnesium –and the two combined– on platelet activation. Using PASII as basis for platelet storage, the addition of 4.5 mmol/L potassium resulted in a pH on Day 7 of 7.13 0.03, versus 6.93 0.04 for PASII alone ( p < 0.001). When 1.5 mmol/L magnesium was added, Day 7 pH was 7.10 0.07 versus 6.98 0.07 for PASII alone ( p < 0.05). Finally, the two combined gave a pH of 7.15 0.10 on Day 7 of storage of the PCs in PASII with potassium and magnesium, versus 6.94 0.05 in PASII alone ( p < 0.05). A notable difference was found in the platelet activation, measured as CD62P expression, that was 23 6% for platelets in PASII with potassium and magnesium versus 50 8% for platelets stored in PASII alone ( p < 0.001). As reference, platelets stored for seven days in plasma had a pH of 7.03 0.06 ( p < 0.05 versus PASII with potassium and magnesium) and a CD62P expression of 35 8% ( p < 0.05). For a similar PAS with potassium and magnesium, Composol, a storage time of at least 12 days could be demonstrated with maintenance of in vitro quality [10]. So, addition of potassium and magnesium inhibits CD62P expression [11], and allows extended platelet storage beyond the currently accepted five or seven days storage intervals. 4. Alternative buffers Though bicarbonate is an excellent buffer, the amount available to prevent acidification of the storage medium may not be sufficient under all conditions. Specifically for pathogen reduction, InterSol (also known as PASIII, Baxter) has been developed [12]. This PAS contains phosphate to maintain neutral pH after the pathogen reduction procedure. However, as demonstrated by Gulliksson et al. [13], phosphate stimulates platelet metabolism. Comparing PASII with PASIII, glucose consumption was 0.06 0.02 mmol/1011 platelets/day in PASII versus 0.08 0.02 mmol/1011 platelets/day for PASIII ( p < 0.001). Lactate production showed the same effect: 0.11 0.03 versus 0.14 0.04 mmol/1011 platelets/day, respectively ( p < 0.001), for PASII and PASIII. Despite this effect, pH remained almost unaffected, 6.97 0.15 in PASII versus 6.94 0.07 for PASIII (not significant), due to the strong buffering capacity of phosphate.
A PAS containing phosphate as well as potassium and magnesium showed possibilities for storage up to 16 days. In a pilot study evaluating storage of platelets in SSP+ (MacoPharma, Toircoing, France), pH was still 7.19 0.06 on Day 16 [14]. Other in vitro markers also indicated good in vitro quality on Day 16, with a CD62P expression of 26 6%, PS exposure of 24 4% and a hypotonic shock-response of 50 15%. In recent studies, bicarbonate is being reconsidered as buffer. Sweeney et al. [15] used bicarbonate pills in a platelet storage container in combination with a glucose-containing PAS with a pH of about 5.2, GASP-BIC. Using PRP-derived platelet concentrates, pH was neutral at 7.07 0.15 on the day of production, compared to 7.45 0.04 for platelets stored in plasma ( p < 0.01). At Day 7 of storage, pH was well maintained at 7.27 0.24 for the bicarbonate-containing PAS, versus 7.19 0.09 for plasma (not significant). Further, CD62P expression was 6 4% for platelets in this PAS, versus 18 10% in plasma ( p < 0.01). Bicarbonate was still present on Day 7:8 1 mmol/L for the PAS and 8 2 mmol/L for the PCs in plasma (not significant). 5. Further developments Instead of using PAS simply as a suspension medium for platelets, nowadays PAS can be considered as a ‘designer solution’ where ingredients can be added to specifically influence certain characteristics of platelet storage. A possible candidate for further development in this respect is L–carnitine. Sweeney et al. showed [16], for platelet concentrates in plasma, that the addition of 5 mmol/L L–carnitine considerably affected storage characteristics. At day 10 of storage, pH was 6.64 0.20 for PCs with L–carnitine, versus 6.42 0.30 for those without ( p < 0.01). This is probably due to inhibition of platelet metabolism by L–carnitine [17], giving lower lactic acid formation. Because L–carnitine has been associated with inhibition of apoptosis by regulating the activity of caspases [18], further studies are warranted. As early as 1996, Klinger et al. [19] demonstrated that at least 30% plasma was required to maintain in vitro quality for platelets stored in PAS. A study by the BEST Collaborative [20] showed a deleterious effect on in vitro measures already if the residual plasma content was lowered from 30 to 20%. Recently, Cauwenberghs et al. [21] showed that washing platelets with a plasma-free buffer rapidly resulted in unresponsiveness to ADP stimulation. She elucidated that ATP- and ADP-degrading activities in plasma preserved the purinergic receptor activities of stored platelets. She was able to show that a substantial part of the ATP-degrading activity in plasma could be inhibited by treatment with anti-CD39 antibody. Although application of an antibody in a PAS is probably difficult to realize, possibly other ways can be found to establish the same effect. If a feasible solution can been found, the residual plasma content may be lowered to less than 5%. Such low percentages residual plasma would decrease adverse effects in patients, notably allergic reactions. In addition, the risk of TRALI would decrease, and AB0-incompatible platelet transfusions could be given easier as no AB0-antibodies are present in the storage medium.
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6. Clinical evaluation of PAS Many studies with PAS only report in vitro data. Although these studies are valuable in the development of new PASs, a follow-up with clinical studies is necessary. So far, only limited clinical evaluations have been published that studied the effectiveness of platelets stored in PASs that were developed in the late 1990s. More recently-developed PASs were specifically investigated only in combination with pathogen reduction technologies. Many of these clinical studies targeted at pathogen-reduction used ‘‘routinelyproduced’’ platelet concentrates as control groups. These, however, are not necessarily the best control group. More often than not, these control groups consisted of platelets stored in PASII, which are known to give lower CCIs [6–8]. As Murphy pointed out [22], this may result in a ‘‘creeping inferiority’’ in the quality of PCs. Therefore, transfusions with fresh PCs in plasma should be included in some form in clinical evaluations of new platelet products in additive solution, to allow fair comparison and to prevent this downward creep. In summary, PASs seem to be an excellent replacement for part of the plasma when producing PCs. Extended storage with maintenance of in vitro quality seems to be feasible. Also, further reduction of the plasma content to below 5% seems to be within reach. Further in vitro studies with respect to the composition of PASs are warranted, and PCs produced with and stored in these newly developed PASs should be evaluated in clinical studies with the right control groups to substantiate in vitro results. References [1] Holme S, Heaton WA, Courtright M. Improved in vivo and in vitro viability of platelet concentrates stored for seven days in a platelet additive solution. Br J Haematol 1987;66:233–8. [2] Rock G, White J, Labow R. Storage of platelets in balanced salt solutions: a simple platelet storage medium. Transfusion 1991;31:21–5. [3] Murphy S. Platelets from pooled buffy coats: an update. Transfusion 2005;45:634–9. [4] Gulliksson H, Eriksson L, Högman CF, Payrat JM. Buffy-coat-derived platelet concentrates prepared from half-strength citrate CPD and CPD whole-blood units. Comparison between three additive solutions: in vitro studies. Vox Sang 1995;68:152–9. [5] Bertolini F, Murphy S, Rebulla P, Sirchia G. Role of acetate during platelet storage in a synthetic medium. Transfusion 1992;32:152–6.
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[6] Turner VS, Mitchell SG, Hawker RJ. More on the comparison of PlasmaLyte A and PAS 2 as platelet additive solutions. Transfusion 1996;36: 1033–4 (Letter). [7] De Wildt-Eggen J, Nauta S, Schrijver JG, van Marwijk Kooy M, Bins M, van Prooijen HC. Reactions and platelet increments after transfusion of platelet concentrates in plasma or an additive solution: a prospective, randomized study. Transfusion 2000;40:398–403. [8] Kerkhoffs JL, Eikenboom JC, Schipperus MS, van Wordragen-Vlaswinkel RJ, Brand R, Harvey MS, et al. A multicenter randomized study of the efficacy of transfusions with platelets stored in platelet additive solution II versus plasma. Blood 2006;108:3210–5. [9] De Wildt-Eggen J, Schrijver JG, Bins M, Gulliksson H. Storage of platelets in additive solutions: effects of magnesium and/or potassium. Transfusion 2002;42:76–80. [10] Van der Meer PF, Pietersz RN, Reesink HW. Storage of platelets in additive solution for up to 12 days with maintenance of good in-vitro quality. Transfusion 2004;44:1204–11. [11] Gawaz M, Ott I, Reininger AJ, Neumann FJ. Effects of magnesium on platelet aggregation and adhesion. Magnesium modulates surface expression of glycoproteins on platelets in vitro and ex vivo. Thromb Haemost 1994;72:912–8. [12] Lin L, Dikeman R, Molini B, Lukehart SA, Lane R, Dupuis K, et al. Photochemical treatment of platelet concentrates with amotosalen and long-wavelength ultraviolet light inactivates a broad spectrum of pathogenic bacteria. Transfusion 2004;44:1496–504. [13] Gulliksson H, Larsson S, Kumlien G, Shanwell A. Storage of platelets in additive solutions: effects of phosphate. Vox Sang 2000;78:176–84. [14] Van der meer PF, Eijzenga-Demmendal M, Pietersz RN. Platelets stored at 4 8C, an evaluation of platelets in SSP+ additive solution. Transfusion 2007;47(Suppl.):84A (Abstract). [15] Sweeney J, Kouttab N, Holme S, Kurtis J, Cheves T, Nelson E. Storage of platelet-rich plasma-derived platelet concentrate pools in plasma and additive solution. Transfusion 2006;46:835–40. [16] Sweeney JD, Blair AJ, Cheves TA, Dottori S, Arduini A. L–carnitine decreases glycolysis in liquid-stored platelets. Transfusion 2000;40:1313–9. [17] Sweeney JD, Arduini A. L–carnitine and its possible role in red cell and platelet storage. Transfus Med Rev 2004;18:58–65. [18] Mutomba MC, Yuan H, Konyavko M, Adachi S, Yokoyama CB, Esser V, et al. Regulation of the activity of caspases by L–carnitine and palmitoylcarnitine. FEBS Lett 2000;478:19–25. [19] Klinger MH, Josch M, Klüter H. Platelets stored in a glucose-free additive solution or in autologous plasma–an ultrastructural and morphometric evaluation. Vox Sang 1996;71:13–20. [20] Gulliksson H, AuBuchon JP, Cardigan R, van der Meer PF, Murphy S, Prowse C, et al. Storage of platelets in additive solutions: a multicentre study of the in vitro effects of potassium and magnesium. Vox Sang 2003;85:199–205. [21] Cauwenberghs S, Feijge MA, Hageman G, Hoylaerts M, Akkerman JW, Curvers J, et al. Plasma ectonucleotidases prevent desensitization of purinergic receptors in stored platelets: importance for platelet activity during thrombus formation. Transfusion 2006;46:1018–28. [22] Murphy S. Radiolabeling of PLTs to assess viability: a proposal for a standard. Transfusion 2004;44:131–3.