Chapter 5
Platelet Products*
Mary Beth Callan1 and Kimberly Marryott2
1Department of Clinical Studies – Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
2Matthew J. Ryan Veterinary Hospital, University of Pennsylvania, Philadelphia, Pennsylvania, USA
*Reproduced and modified with permission from Wiley: Callan, M.B., Appleman, E.H., and Sachais, B.S. (2009) Canine platelet transfusions. Journal of Veterinary Emergency and Critical Care 19, 401–415.
Introduction
Platelet transfusion therapy poses many challenges in veterinary clinical practice. While packed red blood cells (PRBCs) and fresh frozen plasma (FFP) constitute the majority of transfusions administered to dogs and cats in hospitals with ready access to blood components, platelet transfusions can be life-saving in some situations. The lack of a readily available blood donor, the short shelf life of fresh platelet products (fresh whole blood [FWB], platelet-rich plasma [PRP], or platelet concentrate [PC]), and the inability to administer a sufficient number of platelets to meet a patient’s transfusion needs are the major difficulties encountered, even in veterinary institutions with well-established blood bank services. Stored platelet products, including cryopreserved and lyophilized canine platelets, offer the advantage of immediate access when fresh platelets are not available for transfusion.
Indications for platelet transfusions
Thrombocytopenia and thrombopathia
Platelet transfusions are indicated in the management of uncontrolled or life-threatening bleeding due to severe thrombocytopenia or thrombopathia. In clinical practice, immune-mediated thrombocytopenia (IMT) is the most common cause of severe thrombocytopenia in dogs. Blood transfusions might be required in dogs with IMT experiencing severe mucosal surface bleeding, most commonly into the gastrointestinal (GI) tract. In such cases, PRBC transfusions are indicated to provide additional oxygen-carrying support. Platelet transfusions are uncommonly administered to dogs with IMT due to the belief that transfused platelets are rapidly destroyed following administration. However, in dogs with IMT experiencing uncontrolled or life-threatening bleeding (e.g., suspected bleeding into the brain or lungs), platelet transfusions can provide short-term hemostasis despite a negligible increase in platelet count post transfusion. In addition, bleeding associated with hereditary thrombopathias, such as platelet procoagulant deficiency (Scott syndrome) in German Shepherds, can be severe and require platelet transfusions to achieve hemostasis (Figure 5.1). Prophylactic platelet transfusions can be considered in dogs with hereditary thrombopathias and a known bleeding tendency before surgery, with compatible PRBCs or FWB available in the event of excessive bleeding.
Figure 5.1 German Shepherd with severe epistaxis due to platelet procoagulant deficiency (Scott syndrome) that required multiple fresh platelet transfusions to control bleeding.
Massive transfusion
Another potential indication for administration of platelets is massive transfusion, previously defined in dogs as transfusion of a volume of whole blood (WB) or blood components that is greater than the patient’s estimated blood volume within a 24-hour period or replacement of half the patient’s estimated blood volume within 3 hours (Jutkowitz et al. 2002). Many proposed definitions for massive transfusion exist in the human literature, with a recent pediatric study using the transfusion of >40 mL/kg of any blood products within a 24-hour period (Neff et al. 2015). Human trauma resuscitation protocols previously emphasized the use of crystalloids and PRBCs to improve cardiac output and oxygen delivery, with the use of FFP and platelets reserved for patients with persistent hypotension unresponsive to crystalloid infusion, transfusion of >6 units PRBCs, documented abnormal coagulation laboratory parameters, or obvious microvascular bleeding (Holcomb et al. 2008). However, a multicenter prospective cohort study of human patients with hemorrhagic shock from blunt trauma found that in those patients requiring massive transfusion, crystalloid resuscitation in a ratio greater than 1.5:1 per unit of PRBCs transfused was independently associated with a higher risk of multiple organ failure, acute respiratory distress syndrome, and abdominal compartment syndrome (Neal et al. 2012). Furthermore, prospective and retrospective studies of civilian and military trauma, respectively, have documented improved outcomes in massively transfused patients receiving plasma and platelet transfusions earlier and at a lower ratio to PRBC transfusions (Holcomb et al. 2008; Brown et al. 2012; Pidcoke et al. 2012). Although the ideal ratio of blood products for massive transfusion in humans has not yet been clearly defined, clinical practice guidelines have suggested a 1:1:1 ratio of FFP:PRBC:platelet units in an effort to provide a physiologically and hemostatically balanced resuscitation (Pidcoke et al. 2012). Similar massive transfusion guidelines have not yet been evaluated in veterinary patients.
Preparation
While human blood banks maintain PC in their inventories, FWB is the only readily available platelet-containing product in many veterinary practices. Although there are few clinical indications for FWB, it might be appropriate in the management of dogs and cats with anemia and bleeding due to thrombocytopenia or thrombopathia or in those requiring massive transfusion. Whereas the majority of human PC is prepared from single donors by apheresis (selective removal of platelets from the donor’s blood via an automated cell separator with return of red blood cells (RBCs) and plasma to the donor), canine PRP and PC generally are prepared from FWB on an as-needed basis by select veterinary blood banks. Due to the limited need, as well as technical challenges associated with small volume blood collection bags for cats, feline PRP and PC are prepared infrequently.
Whole blood-derived platelets
Human
In human blood banks in North America, the standard method for platelet component production from FWB is the PRP-derived PC method, whereas in Europe the buffy coat-derived PC method is used. In the former method, FWB is stored at 22°C for up to 8 hours before “soft spin” centrifugation (2000 g for 3 minutes) to produce PRP, which can then be leukoreduced through an integrated filter as the PRP is expressed off the RBC product (Vasallo and Murphy 2006; Fung 2014). The leukoreduced PRP then undergoes “hard spin” centrifugation (5000 g for 5 minutes) to produce a single unit of PC, with the potential to pool 4–6 PC units just before transfusion; single PCs are stored for up to 5 days, whereas pooled PCs may be stored for up to 4 hours or 5 days if using an open or closed system, respectively, for pooling of units (Fung 2014). In the buffy coat-derived PC method, butanediol plates within the storage containers rapidly cool and hold WB at 22°C for up to 24 hours before processing; the WB undergoes “hard spin” centrifugation to concentrate 90–95% of the platelets along with the white blood cells (WBCs) in the buffy coat (Vasallo and Murphy 2006; Fung 2014). Typically four to six buffy coats are pooled, “soft centrifuged” to remove contaminating WBCs and RBCs, and then passed through a leukoreduction filter. The platelet yields are reported to be similar for both methods. The proposed benefits of the buffy coat-derived PC method include the convenience of storing WB for up to 1 day before processing, pre-storage pooling of platelets, and greater plasma yield (∼30–75 mL/U of whole blood) (Vasallo and Murphy 2006). As part of the quality control of WB-derived PC preparation, the American Association of Blood Banks (AABB) requires that at least 75% of the units tested must contain ≥5.5 × 1010 platelets and at least 90% of the units sampled have a plasma pH ≥ 6.2 at the end of the allowable storage time. In addition, pre-storage leukocyte-reduced platelets derived from filtration of PRP must contain <8.3 × 105 residual leukocytes per unit to be labeled as leukocyte reduced (Fung 2014).
While the United States Food and Drug Administration (USFDA) requires that PRP-derived PC be prepared from FWB within 8 hours of collection, a few studies have evaluated PCs prepared from WB stored for 20–24 hours at 22°C, since overnight storage would be a convenient option for blood banks. The longer storage of WB resulted in platelet yields well above the FDA requirements of 5.5 × 1010 platelets per unit (i.e., overall mean 8.78 × 1010 ± 2.37 × 1010) (Slichter et al. 2012a). In addition, the in vitro quality (e.g., hypotonic shock response, expression of activation marker CD62P) of PRP-derived PCs prepared from WB that had been stored overnight was at least of equal quality as that from freshly processed WB (Van der Meer et al. 2011).
Canine
Similar to the human PRP-derived PC method, canine PRP can be harvested from FWB after “soft spin” (1000 g for 4 minutes) centrifugation (Figure 5.2), with the supernatant PRP then centrifuged further using a “hard spin” (2000 g for 10 minutes) to produce PC (Box 5.1). In a study evaluating preparation of canine PCs from FWB using this method, the mean platelet yield was 8 × 1010 per PC unit, with 80% of the units containing >5.5 × 1010 platelets and 24% of the units having a platelet yield >1 × 1011 (Abrams-Ogg et al. 1993). The mean platelet yield from FWB to PC was 74%, indicating that approximately 25% of platelets in a unit of FWB are lost during processing. The leukocyte content of the PC units ranged from 1.0 × 108 to 2.3 × 109 WBCs, and the hematocrit (HCT) ranged from 0.1% to 26.2%, with 62% of the units having a HCT > 1%. In a subsequent study of canine PCs by the same researchers, the HCT of all PC units was reduced to <1% by stopping expression of PRP when the RBC-plasma interface was 1 cm from the top of the bag (Allyson et al. 1997).
Figure 5.2 Separation of platelet-rich plasma. a. Blood collection bag on plasma extractor after soft spin centrifugation. b. Supernatant platelet-rich plasma expressed into satellite bag. c. Platelet-rich plasma and packed red blood cells after separation.
There are many centrifugation protocols described for the preparation of canine PRP that are described elsewhere in this textbook (see Chapter 17). It is recommended to calibrate each centrifuge individually to determine the shortest time and lowest speed that results in the highest yield of platelets. Preparation of canine PC by a buffy coat protocol has been described, but resulted in platelet yields lower than those reported above for canine PRP-derived PC and less than the AABB standard of >5.5 × 1010 platelets per unit (Hoareau et al. 2014).
Feline
While there is a published report of platelet transfusions administered to research cats with Chediak–Higashi syndrome in which the PRP was prepared from feline FWB in polypropylene tubes centrifuged at 150 g for 10 minutes (Cowles et al. 1992), information regarding preparation of feline PRP in blood collection bags is lacking. However, feline PRP has been successfully prepared using a small double-bag collection system with centrifugation at 1000 g for 3 minutes (K. Jane Wardrop, personal communication 2014). Due to the small size of feline collection bags, the use of inserts in the centrifuge buckets is necessary to prevent collapse of the bag during centrifugation. Further details regarding processing of feline blood products can be found elsewhere in this textbook (see Chapter 17).
Apheresis platelets
Human
An alternative approach to preparation of PC from FWB is plateletpheresis. During the apheresis procedure, blood is removed from the donor, anticoagulated with citrate (typically, acid-citrate-dextrose formula A [ACD-A]) in the extracorporeal circuit, and separated into components by centrifugation, allowing production of a PC, while the other blood components are returned to the donor. The advantages of PC prepared by apheresis, in comparison to PRP or PC prepared from a unit of FWB, are greater platelet yield (typically 3–4.5 × 1011 versus <1 × 1011) and negligible RBC and WBC contamination. Transfusion of apheresis-derived platelets also decreases recipient exposure to donors, as pooling of products is not required. Plateletpheresis is generally well tolerated by human donors, though decreased levels of ionized calcium and magnesium occur commonly due to citrate administration and cause clinical signs including paresthesias, chills, headache, lightheadedness, carpopedal spasm, nausea, vomiting, chest tightness, and cramping (Bolan et al. 2001).
Canine
Automated blood cell separators that have been used for canine plateletpheresis include the AS104 (Fresenius AG, Hamburg, Germany) (Adamik et al. 1997), MCS Plus (Haemonetics, Braintree, MA) (Williamson and Hale 2007), and COBE Spectra (Caridian BCT, Lakewood, CO) (Callan et al. 2008). In a study evaluating the clinical and clinicopathologic effects of plateletpheresis on 14 healthy donor dogs weighing approximately 20 kg using the COBE Spectra (dual needle, leukocyte reduction system) a high-quality PC (Figure 5.3) was collected from all dogs, with a mean total platelet yield of 3.3 × 1011 platelets in a mean collection volume of 246 mL, similar to the yield and volume collected from adult human donors. The mean donor platelet count decreased by 55% from 356 × 109/L (356,000/μL) to 159 × 109/L (159,000/μL) 2 hours after apheresis but returned to baseline level by day 6.
Figure 5.3 Apheresis platelet concentrate obtained from a 20-kg donor dog using the COBE Spectra. The platelet concentrate contains approximately 3 × 1011 platelets, about the same number of platelets as five whole blood-derived platelet concentrates.
The plateletpheresis procedure was generally well tolerated with no evidence of hypotension. However, ionized hypocalcemia (<1 mmol/L) was measured in 10 of 14 dogs, despite calcium supplementation (0.6–0.8 mL/kg/hour of 10% calcium gluconate as a constant rate intravenous infusion). Associated clinical signs noted in three dogs included intermittent lip licking and agitation, with generalized tremors and sporadic ventricular ectopy also noted in the dog with the lowest ionized calcium concentration (0.76 mmol/L). While plateletpheresis is a feasible option for production of canine PC, citrate-induced hypocalcemia is a potential serious adverse effect. Prophylactic calcium administration is warranted to limit clinical signs of hypocalcemia when plateletpheresis is performed in dogs using high citrate infusion rates (Callan et al. 2008).
Storage
Fresh platelets
Fresh platelet products have a short shelf life. Fresh whole blood can be stored at room temperature (22°C) for up to 8 hours, whereas PRP and PC can be stored at 22°C with continuous gentle agitation (Figure 5.4) for up to 5 days (referred to as liquid-stored platelets beyond day 0) (Fung 2014). The majority of human platelets collected by apheresis in the United States are stored in 100% plasma and ACD-A. However, hyper-concentrated apheresis platelets can be diluted and stored in platelet additive solutions, thereby reducing the risk of allergic transfusion reactions and transfusion-related acute lung injury in which plasma proteins have been implicated (Dumont et al. 2013a; Slichter et al. 2014).
Figure 5.4 Storage of fresh platelet concentrate undergoing continuous agitation on a blood product agitator or “rocker”. (Image courtesy of Marie K. Holowaychuk, DVM, DACVECC.)
The time limitation on storage of platelets at 22°C is primarily related to concerns about potential for rapid proliferation of bacteria at this temperature and a progressive decrease in platelet viability and function over time. During storage, platelets undergo a variety of in vitro changes that are collectively referred to as the “platelet storage lesion”. This lesion is characterized by a change in platelet shape from discoid to spherical, the generation of lactate from glycolysis with an associated decrease in pH, the release of granule contents, a decrease in various measurements of in vitro platelet function, and reduction in post-transfusion platelet recovery (percentage of transfused platelets that survive in the recipient) and survival (lifespan of transfused platelets) (Fung 2014). The USFDA’s criteria for determining maximum duration of platelet storage include autologous stored platelet recoveries and survivals that are ≥66% and ≥58% of each donor’s fresh platelet collection, respectively (Slichter et al. 2014).
There has been much interest in both human and veterinary transfusion medicine to develop alternative storage options (i.e., cold storage, cryopreservation, and lyophilization) that would increase the shelf life and availability of platelet products in clinical practice (Table 5.1). However, current conditions for storage of human PCs for transfusion remain at 22°C under continuous gentle agitation.
Table 5.1 Characteristics of platelet concentrates under various storage conditions
| Fresh platelets (or liquid-stored platelets) | Chilled platelets | Cryopreserved platelets | Lyophilized platelets | |
| Storage conditions | 22°C with continuous gentle agitation | 4°C | 6% DMSO, –80°C (≤–20°C in practice) | –80°C or 4°C |
| Shelf life | 5 days | N/D | 6 months (–20°C) to 1 year (–80°C) | Several years (–80°C) or 2 years (4°C) |
| Advantages | Optimal post-transfusion platelet recovery (dog, 80%), survival (dog, half-life 3.8 days), and function | Decreased risk of bacterial proliferation during storage Improved retention of in vitro function during storage | Long-term storage Immediate availability High levels of expression of phosphatidylserine, potentially promoting thrombin generation in dogs with platelet procoagulant deficiency | Long-term storage Immediate availability Sterility (result of paraformaldehyde stabilization) |
| Disadvantages | Short shelf life Limited availability Risk of bacterial proliferation during room temperature storage | Rapidly cleared from circulation (in humans and mice, with and without modification) | Reduced post-transfusion platelet recovery (dog, 49%) and half-life (dog, 2 days) Impaired in vitro function, though evidence of hemostatic efficacy in vivo | Short in vivo lifespan (minutes in rats and baboons) Use limited to control of active hemorrhage |
Chilled platelets
Human
Refrigeration of PC could reduce the problem associated with proliferation of contaminated bacteria at room temperature. However, as early as 1969 there was strong evidence that chilled storage of human platelets had a deleterious effect on platelet viability. Storage of human PRP at 4°C for as little as 18 hours with subsequent infusion into the same donor resulted in a markedly reduced platelet survival with a half-life of 1.3 days in comparison to 3.9 days for platelets stored at 22°C for 18 hours (Murphy and Gardner 1969). Following this discovery, it became standard procedure for human PRP to be stored at 22°C. The mechanism responsible for rapid clearance from the circulation of refrigerated platelets was not elucidated until more than 30 years later. Chilling of human and mouse platelets clusters their von Willebrand factor (VWF) receptors (GPIbαβ-IX complex) on the platelet surface, with the clustered GPIbα recognized by hepatic macrophages, which then ingest the platelets (Hoffmeister et al. 2003). Attempts to block the interaction between GPIbα and hepatic macrophages via galactosylation of exposed β-N-acetylglucosamine residues failed to prevent the accelerated clearance of human platelets stored for 48 hours at 4°C following autologous transfusions, with a mean platelet survival of 2.2 days compared with 6.9 days for fresh platelets (Wandall et al. 2008).
Refrigerated human platelets, whether galactosylated or untreated, either in WB or PC, retain in vitro function better than platelets stored at 22°C (Babic et al. 2007). Furthermore, data from a randomized clinical trial of children undergoing cardiac surgery suggest that platelets in WB stored at 4°C for 24–48 hours are more hemostatically active than platelets stored at 22°C and combined with RBCs and plasma (Manno et al. 1991). The question of whether human blood banks should maintain a stock of refrigerated platelets is controversial, and recently the debate has been revived. While refrigerated platelets would not be ideal for prophylactic platelet transfusions given their short in vivo survival, some argue that the decreased risk of bacterial proliferation and enhanced hemostatic activity of chilled platelets would be beneficial in patients with acute hemorrhage (Pidcoke et al. 2014).
Canine
To date, there are no published reports on in vivo survival of chilled canine platelets. In a study of storage of canine PCs at 22 and 4°C, it was noted that platelets stored at 22°C lost their ability to aggregate in vitro in response to ADP and collagen after 4 days, whereas platelets stored at 4°C did not lose their ability to aggregate before 8 days of storage (Klein et al. 1999). Similar loss of platelet function as assessed by resonance thrombography was noted over the same time frame. Consequently, it was concluded that canine PC can be stored for 4 days at 22°C or 8–10 days at 4°C (Klein et al. 1999). In light of the effect of chilling on survival of both murine and human platelets post transfusion, it is reasonable to hypothesize that refrigerated storage of canine PC would also lead to decreased post-transfusion survival. Further investigation of the effects of cold (4°C) storage on the in vivo survival and hemostatic efficacy of canine platelets would be warranted prior to recommending that canine PC be refrigerated.
Cryopreserved platelets
Human
During the past several decades, platelet cryopreservation has been extensively investigated as a means to provide long-term storage and immediate availability of platelet products for transfusion. The AABB, but not yet the USFDA, has approved cryopreservation of human platelets in 5–6% DMSO as an acceptable storage procedure, but platelet cryopreservation is largely considered a research technique. Based on the numerous studies evaluating cryopreservation of human platelets, it is clear that regardless of the methodology, cryopreserved platelets (CPPs) inevitably demonstrate impaired in vitro function and reduced post-transfusion recovery in comparison to fresh platelets (Melaragno et al. 1985; Dumont et al. 2013b). However, it has been documented that assays of in vitro platelet function do not predict platelet function and viability in vivo (Khuri et al. 1999; Slichter et al. 2013). Furthermore, there is clinical evidence that human CPPs are hemostatically effective in vivo. In a prospective randomized study of human patients undergoing cardiopulmonary bypass surgery, patients that received CPPs compared to room temperature-stored PC experienced reduced blood loss and need for blood products in the postoperative period (Khuri et al. 1999).
Human platelets cryopreserved in 6% DMSO traditionally required a post-thaw washing step to remove the DMSO before transfusion to decrease the risk of adverse effects from the DMSO (e.g., nausea, fever, hypo- or hypertension). However, the technique has been modified so that fresh platelets are treated with 6% DMSO, concentrated via centrifugation to remove the supernatant DMSO, frozen at –80°C, thawed, and diluted with 0.9% NaCl (Valeri et al
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