Novel treatment modalities: new platelet preparations and subsititutes

Despite advances in the safety, processing and storage of conventional liquid-stored allogeneic platelet concentrates, there are still several drawbacks to standard platelet concentrates. Efforts to overcome these shortcomings have been undertaken by both the academic and commercial sectors, resulting in an array of novel platelet products and platelet substitutes, all at various stages of development. This review will examine the development of new platelet products, synthetic platelet substitutes, new storage methods for platelets and pathogen inactivation methods for platelet concentrates. A brief account of platelet transfusion history may help to place this review within the context of past and current platelet transfusion practices. Before the 1950s, fresh whole blood or platelet-rich plasma transfusions were the only therapeutic agents available to treat thrombocytopenic bleeding. These treatments were cumbersome and their effectiveness was limited by the risk of volume overload. Methods for separating platelets using centrifugation were developed in the 1950s and 1960s, allowing platelet concentrates to be given; however, the shelf-life of the early platelet concentrates was only a few hours. In an unsuccessful attempt to prolong the storage of platelets, freezing and lyophilization methods were investigated during this era. Initially, platelets were stored at 4°C with a resultant shelf-life of only 24 h for haemostatically active platelets. In the 1970s, room temperature-stored platelets were shown to have greater haemostatic efficacy and survival than platelets stored at 4°C. With the improvement to storage containers and conditions, platelet concentrates could be stored for up to 5 d at 22°C by the mid 1980s. In 1985, storage at room temperature was extended to 7 d; however, concerns about the high reaction rate and potential extent of bacterial proliferation during 7 d storage forced regulators to curtail the shelf-life to 5 d. At the present time, platelets for therapeutic use can be prepared from either units of donor whole blood or by apheresis. Whole blood-derived platelets can be prepared using the platelet-rich plasma (PRP) technique (the main one currently in use in North America) or by the buffy coat (BC) technique (the main method used in Europe). The differences between these two approaches have been discussed in detail by Murphy et al (1996). In the PRP approach, the whole blood is collected into an anticoagulant–nutrient solution, then centrifuged to separate the PRP. The PRP is centrifuged again to prepare the platelet concentrate (PC), which contains 60–75% of the platelets present in the whole blood unit from which it is derived. Presently, PRP-platelet concentrates are stored in approximately 40–50 ml of plasma for up to 5 d at 22°C, with gentle continuous agitation, in specialized plastic bags that are permeable to oxygen and carbon dioxide (Murphy, 1985; Moroff & Holme, 1991). Typically, an adult dose consists of the PCs from five donors. In contrast to the PRP method, the BC method, which evolved in Europe, involves the removal of the BC after an initial ‘hard’ centrifugation of a whole blood unit. The BC aliquot, which also contains the platelets, is then removed and 4–6 units pooled. This pool is then subjected to a soft spin to differentially concentrate the platelets and the leucocytes. The leucocytes are then removed and discarded and the PC made as per the PRP approach and stored in a 200–300 ml volume for up to 5 d at 22°C. An alternate method of PC preparation is apheresis. In the apheresis methods, whole blood drawn from a donor is mixed immediately with an anticoagulant/nutrient mixture and centrifuged, during which time the components are separated according to density. The various components (buffy coat, platelets) are recognized by optical sensors that cause the platelets and/or other components to be directed into separate containers. Those components not required (i.e. red blood cells) are returned to the donor. The different apheresis equipment manufacturers have developed a variety of approaches to isolating the platelet fractions including: (i) plasma recirculation and elutriation; (ii) discontinuous flow separation; and (iii) continuous flow separation using special chambers. Apheresed platelets from a single donor are stored in approximately 300 ml of plasma and are not pooled before transfusion because they contain approximately the equivalent of 5 units of whole blood PRP-derived platelets. Transmission of infection is minimized by excluding high-risk individuals from the donor pool and by the serological testing of donors. In recent years, the recognition that leucocytes found in PC may result in undesirable biological and clinical effects (Bordin et al, 1994) has led to the institution of pre-storage leucoreduction in many jurisdictions. Despite improvements in the production of 22°C liquid stored platelets, conventional PCs have several drawbacks (Table I). Recipients continue to be at risk of febrile non-haemolytic transfusion reactions (Heddle et al, 1993), the transmission of bacterial (Blajchman, 1998a), viral and protozoan infections, alloimmunization resulting in refractoriness to future platelet transfusions (Slichter, 1998; Novotny, 1999) and graft-versus-host disease (Orlin & Ellis, 1997). The potential for transfusion-associated immunosuppression has also been a concern (Blajchman, 1998b). Shortages in supply occur frequently, and the stockpiling of platelets is not feasible because of the short shelf-life of conventional platelet concentrates. This often results in wastage owing to the non use of time-expired platelet products, inevitable under current transfusion medicine practices. The decline in the donor pool and increase in platelet use over the past decade compounds the problem. These drawbacks have led to efforts to minimize the exposure of recipients to allogeneic blood products and to develop safe and effective platelet products and substitutes with longer shelf-lives. Modified platelet products and substitutes should function haemostatically as live platelets do without causing pathological thrombosis or a consumptive coagulopathy. A platelet substitute should not transmit infection, nor should it be immunogenic or cause reticuloendothelial blockade. Ideally, novel platelet products and platelet substitutes should have a long duration of action to allow long-dosing intervals. Preferably, they should have simple storage requirements (such as room temperature), a long shelf-life and should be easy to administer (off-the-shelf administration or reconstitution in conventional crystalloid solutions and administration without washing) (Table II). Although the primary goal of this research has been to develop products that are safe and haemostatically effective, live platelets have many complex functions other than clot formation. These include the modulation of fibrinolysis, inflammation, vascular tone and cellular growth through substances released from cytoplasmic granules or direct interaction with endothelial cells, leucocytes and macrophages. Many of these functions are poorly understood and serve as a reminder of the complexity of the cell that researchers seek to mimic. Table III lists the novel platelet products and substitutes that are currently at various stages of development. Presently, frozen platelets are the only accepted alternative to conventional platelet concentrates. However, cryopreservation is cumbersome and expensive, with the result that freezing has generally been used primarily for storing autologous platelets for transfusion in alloimmunized patients with acute leukaemia. Moreover, for most clinical indications, cryopreserved platelets do not offer advantages over conventional platelet concentrates and are more laborious and costly to prepare. Moreover, most hospitals lack the facilities and trained personnel needed to prepare, store and process frozen platelets. Platelets frozen in 5–6% dimethylsulphoxide (DMSO) can be stored for years at −80°C, and currently represent the standard for long-term cryopreservation. Washing is required to remove excess DMSO before transfusion. Recovery post-thaw is approximately 75% and after transfusion into normal volunteers, in vivo platelet recovery at 1 h is approximately 33% with a platelet survival thereafter of about 8 d (Melaragno et al, 1985). Cryopreserved platelets acquire a variety of morphological and functional defects in vitro (Spector et al, 1977; Shepherd et al, 1984; Melaragno et al, 1985) but are haemostatically effective in vivo (Schiffer et al, 1978; Melaragno et al, 1985). In one recent study, cryopreserved platelets were reported to be more effective in decreasing blood loss in cardiopulmonary bypass surgery than standard liquid stored platelets (Khuri et al, 1999). Under most circumstances, 6% DMSO frozen platelets do not offer advantages over conventional liquid-stored platelets, and are significantly more laborious to prepare. For these reasons, the clinical use of frozen platelets is not widespread. In recent years, the addition of a cryoprotectant consisting of amiloride, adenosine and sodium nitroprusside (ThromboSol™, LifeCell, The Woodlands, USA) has been reported to decrease the requirement of DMSO from 6% to 2% with improved post-thaw in vitro platelet recovery and function (Currie et al, 1998). These second messenger effectors inhibit cold-induced platelet activation pathways, thereby blunting the detrimental effects of cold storage. In vivo recovery of platelets cryopreserved in ThromboSol™ and 2% DMSO was shown to be superior to those frozen in 6% DMSO with normal platelet survival (Currie et al, 1999). In patients with chemotherapy-induced thrombocytopenia, a 1 h mean corrected platelet count increment of 9·2 × 109/l was achieved when controlled rate freezing was used (Pedrazzoli et al, 2000). The storage of liquid-suspended platelets at 4°C retards bacterial growth, but platelets stored in the cold in plasma for 24 h or more have a shortened survival and are less effective haemostatically than platelets stored at 22°C (Murphy & Gardner, 1969; Slichter & Harker, 1976; Filip & Aster, 1978). Several strategies have been used in an attempt to preserve platelet morphology and function. However, the pathophysiology of the cold-induced platelet storage lesion remains incompletely understood (Vostal & Mondoro, 1997). One approach to preventing cold activation of platelets has been to inhibit cytoskeleton actin assembly, the defect implicated in the disc-to-sphere-shape change associated with platelet chilling. Platelets cooled to 4°C in the presence of cytochalasin B and a cytoplasmic Ca2+ chelator remain discoid and responsive to thrombin when Ca2+ is added to overcome the chelator (Winokur & Hartwig, 1995). In addition to its use as a cryoprotectant, ThromboSol™ has been used as an additive solution to permit cold storage of liquid-suspended platelets. Platelets stored at 4°C in this fashion have greater retention of in vitro function with less evidence of cold-induced activation despite loss of discoid shape (Connor et al, 1996; Rivera et al, 1999; Lozano et al, 2000). However, in vivo haemostatic function and survival have not yet been reported. A different approach involves the use of antifreeze glycoproteins (AFGPs) found in the circulation of polar fishes. Although cold-induced platelet shape change and activation are inhibited by AFGPs (Tablin et al, 1996), haemostatic activity in vivo has yet to be reported. Photochemical treatment of liquid-stored platelets with psoralen followed by illumination with long-wavelength ultraviolet radiation (UVA 320–400 nm) is a pathogen inactivation strategy that targets the nucleic acids. This technology offers a potentially complementary approach to that of serological testing of donors for various infectious agents (Corash et al, 2000). Psoralens are tricyclic compounds that react with helical DNA and RNA in a two-step mechanism. The psoralen molecule first intercalates within a double helical region of nucleic acid. When exposed to UVA light, the psoralen becomes covalently bound to pyrimidine bases, forming cross-links between strands of nucleic acid et al, 1985). and occur the resulting in the inactivation of and Photochemical treatment using the synthetic psoralen a variety of and added to platelet concentrates, without in vitro platelet function et al, 1997). production during platelet storage and transfusion-associated in an have been reported to be by treatment with et al, 1999; et al, that inactivation also results of a clinical platelet concentrates with platelets in thrombocytopenic patients were reported et al, 2000). The of acute transfusion reactions and platelet refractoriness were between the Although the between platelet transfusions was not found to significantly between the transfusion of platelets was associated with a platelet corrected count increment at 24 are of platelet that during the activation or of platelets during platelet storage and can be found in platelet concentrates et al, et al, et al, fresh frozen plasma and et al, activity & et al, to vascular and platelet et al, haemostatic as platelets, they a strategy for the development of a platelet However, the of platelet were not effective haemostatic agents and in et al, This of research was for in thrombocytopenic evidence of their haemostatic efficacy without et al, a 1 platelet as platelet has been developed USA) et al, are prepared from blood platelet and are removed using centrifugation and the platelets are by the preparation is at for h to followed by and The is in and and then a that is for over years at 4°C. platelet of with a of approximately and are of and The of and is to that of platelets, and for several cytoplasmic and components are not is present in and to be functional because is et al, but is not in A recent using an vivo vascular flow evidence that activity at of vascular under of and was also but only under of et al, 2000). In shortened the time in thrombocytopenic for at h after however, by 24 h this haemostatic was longer have not pathological in the et al, In normal volunteers, of were and or evidence of was reported et al, 1997). In a of a clinical study, of thrombocytopenic patients or of a of at one of two to h after however, were also in two of two patients after of et al, 1995). In a patients with platelet of × 109/l and either a single dose of from to or conventional platelet concentrate et al, 1997). or of was in of patients and of five platelets. A III clinical of is In the 1950s, of the effectiveness of platelets in a of thrombocytopenic patients et al, & were followed by controlled in thrombocytopenic that to the haemostatic efficacy of platelets et al, et al, et al, of platelets a long from the a platelet preparation has with more and developed a platelet lyophilization the of and several functional with a of platelets from whole blood et al, platelets are with for platelets, or for then frozen in and at to for The process has also been reported to be and et al, When platelets are to fresh platelets, with to in and by flow et al, et al, that platelets a that was that of fresh platelets, and could of the with fresh platelets. of in vitro that was associated with the platelet but more than fresh platelets, in to the of bound to the platelet et al, A preparation of platelets was of clot et al, 1998). The that is is functional because it is of et al, 1997). platelets in the presence of or & 1978). do not in to or but may in when fresh platelets are also present et al, 1997). The of platelets activity et al, and increase exposed in the vascular et al, 1997). platelets also to exposed but to a extent than fresh platelets et al, et al, 1999). discoid have also been reported in that platelet also The of activity to is by the that a increase in the of activation in the platelets in the Moreover, these platelets were found to et al, 1999). of and in platelets was that are et al, 2000). platelets to be of than as a or The in vivo effects of platelets have been in a variety of et al, et al, in that were with platelets that this can be in the circulation and not a consumptive coagulopathy. of of the platelets to and into et al, platelets have been shown to be haemostatically effective in thrombocytopenic The thrombocytopenic was used to the in vivo recovery and haemostatic efficacy of platelets & 1997). recovery at 1 h was for platelets, with for fresh platelets. The of 40–50 × platelets per shortened the time in thrombocytopenic from to a mean of platelets) and for fresh platelets et al, platelets also corrected the time associated with cardiopulmonary bypass in a surgery et al, with platelets continue to be alternative However, as as are clinical with platelets are in the Platelets that are of can be in vitro from from normal blood by et al, 1995). this approach will be of use in platelets for transfusion is but an The that platelets with or with were made years & that with platelets to however, this interaction was by the addition of or & found that platelets covalently bound could in with fresh platelets. these the use of autologous with covalently bound to the was as a potential haemostatic in & was to and red by with or to a of per red platelet by or in a fashion that was the during the cell bound by and with were by the treatment in thrombocytopenic were shortened from a mean of to 1 h after with 1 × red The duration of the haemostatic was longer than that after of fresh platelets. Moreover, this was using red cell as few as per red cell & et al used a different than to the red cell they to the In a study, of a were bound the to and their to with platelets was et al, The that the be extended from the for interaction with was to be the of the could be that interaction was the of platelet were to the of red and the was to this platelet substitute et al, platelet and with platelets exposed to These effects could be with a for the platelets et al, not with platelets but to platelets to both in and under of in flow et al, At of was less The process not red cell and An initial of in vivo efficacy that shortened the time in thrombocytopenic et al, but the of a 2% solution of not the time in thrombocytopenic et al, 1997). The that either or platelets the could platelet & & a for the of as potential haemostatic agents in about the of to through the has a two different of are in et al, 1999) are that are to that have been developed for use as an contrast et al, 1997). are by a solution of to is to the under and a that less than 2% of the of the The of the is with than 2% a in excess of et al, 1999). In a thrombocytopenic a single of × shortened the time from a mean of to at after the blood loss from a standard This haemostatic was still at but was longer 8 h after was using a thrombosis and cardiopulmonary was in using thrombocytopenic and normal whole blood in a that the of to endothelial These were found to contain platelets and an interaction of with platelets. an interaction is also by the that the were with platelets and in of the time et al, 1999). in vivo haemostatic results have been reported with a preparation with a mean of using a different process et al, also shortened the time in thrombocytopenic but with a duration of action of at h after a single The of action is poorly understood because the haemostatic of after are longer in the In normal not platelet survival and were not found to be et al, The reconstitution of platelet glycoproteins into was by several in the et al, et al, 1985; & 1985; were used to the of glycoproteins and the of and in platelet A platelet substitute the was by & were by a of platelet and into consisting of or platelet in However, of the blood loss in thrombocytopenic and with platelet storage pool evidence of consumptive was after of into nor was there evidence of pathological thrombosis in & In a recent of or were found to to or under flow conditions, but in vivo have been reported with this preparation et al, 1998). and An of a of and has been to in & However, this only a the time and blood loss in with storage pool disease et al, 1997). Moreover, the of and was associated with et al, 1997). synthetic have been without et al, 1998). This approach has been reported to exposed in thrombocytopenic blood in the novel platelet products and substitutes clinical researchers and regulators are with the of clinical of haemostatic A in the clinical of platelet products and substitutes is the of thrombocytopenic (Vostal et al, 2000). occur in thrombocytopenic patients and using will is to in a standard and and have not been This will to be to permit clinical testing using thrombocytopenic in the of thrombocytopenic bleeding. has been by the red cell in et al, et al, more by the time or blood loss from or & et al, 1999). The when using is that the haemostatic function. and their remains a One of the concerns about novel platelet products and substitutes is their potential for the platelet substitutes, that to agents with which are the is that of pathological thrombosis and consumptive with novel has been the effects of platelet and for platelet products and are for products that and the potential for alloimmunization be the potential of the reticuloendothelial by or platelet substitutes a approach to these is the risk of infection with these the treatment of liquid-stored platelets, the of the novel products and substitutes are A of are associated with platelet refractoriness in The main one is The approach to patients has been to platelets from donors. approach is to the of the platelets to approaches have been used for the (i) and (i) The is using Although from the platelet has been shown to be platelet can be by A review of this has been et al, 1999) and the available that the platelet achieved with the use of platelets is the use of platelets at this time should be and for clinical has been shown to be haemostatically effective in the treatment of in patients with an This has also been used in thrombocytopenic patients and patients with platelet both and the administration of has been shown to result in a in the in thrombocytopenic patients et al, as as in patients with & & 1996; et al, 1999) and patients with platelet associated with a et al, 1998). However, these still be as and clinical be to the efficacy of in the treatment of patients with and platelet function the various strategies for novel platelet products and substitutes have most of these have been Presently, the only accepted alternative to conventional 22°C liquid-stored platelets are frozen platelets, but these are used is that platelet products that are by to the current liquid-stored standard (such as liquid-stored platelets) may clinical use before of the platelet The platelet substitutes and other products more and clinical before their use in patients can be In the there will be thrombocytopenic in which platelet products will be However, the of use for platelet products will change in the future as they have in the Although growth as and which minimize chemotherapy-induced thrombocytopenia, are not in these new advances serve as a reminder that the development and of platelet products and substitutes not a With advances in and there is to that effective and platelet products and substitutes will for clinical use in patients with

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