Platelet storage lesion: An update

Platelets are unique blood cells that play a pivotal role in mediating hemostasis and thrombosis. Over the years, the demand for platelets is steadily rising because of their increased use in patients with thrombocytopenia and platelet function defects. The availability of platelets for transfusion is restricted by the markedly short shelf life of platelets owing to risk of bacterial contamination and storage-related deterioration known as platelet storage lesion (PSL).[1] PSL is best defined as the sum of all the deleterious changes in platelet structure and function that arise from the time the blood is withdrawn from the donor to the time the platelets are transfused to the recipient.[2] The mechanisms responsible for PSL are multifactorial and not clearly understood. Several factors including the methods of collection, processing, storage, and manipulation after collection can result in PSL.[3] These lesions are associated with decreased in vivo platelet recovery, survival, and hemostatic activity after transfusion.[4]

Preservation of platelet structure, composition, and function during preparation and storage is one of the primary goals of transfusion medicine practice.[5] Technical advances in the form of introduction of automated cell separators, optimized centrifugation procedures, improved storage containers, leucoreduction, and platelet additive solutions (PAS) have greatly facilitated the collection and storage of platelets. Despite these advances, PSL is still a matter of concern. Platelets get activated following exposure to foreign surfaces, trauma, low pH, agonists (thrombin, ADP), and shear stress. Upon activation, the platelets lose their discoid morphology and become more spherical with multiple pseudopods. Conformational changes in GPIIb/IIIa complex exposes binding sites for adhesive proteins (fibrinogen, vWF) resulting in platelet aggregates. Activation further stimulates release of granular contents and expression of sequestered membrane proteins on outer surface. Release of these granular contents at the site of injury is indispensable for platelet function as the contents not only mediate the recruitment of leucocytes and platelets, promote clotting but also provide immunity against infection and contribute to wound healing.[6] Moreover, the presence of these contents in storage medium can lead to various transfusion reactions.[7] Expression of negatively charged phospholipids (phosphatidyl serine and phosphatidyl ethanolamine) increases on the outer leaflet of activated platelets, providing a surface for the prothrombinase complex (X-Va), thereby contributing to procoagulant activity.

Exposure to shear stress, as in centrifugation, during component separation and agitation while storage not only activates platelets but can also cause their lysis and calpain activation.[5] Platelet lysis leads to discharge and accumulation of cytosolic lactate dehydrogenase (LDH) and granular contents. Calpain (protease) activation results in degradation of cytoskeletal proteins such as actin, talin, and actin binding protein generating platelet microvesicles.[5] Microvesicle formation leads to decrease in mean platelet volume (MPV) and also contributes to procoagulant activity. Adenosine triphosphate (ATP) depletion occurs as ATP is required for each aspect of platelet function and is generated via oxidative and glycolytic pathways. Glycolysis is upregulated under low oxygen conditions. Lactate produced via glycolysis is buffered primarily by plasma bicarbonate. Progressive lactate production and bicarbonate exhaustion during storage results in deleterious decrease in pH augmenting platelet damage. Platelet apoptosis occurring simultaneously during storage further contributes to loss of mitochondrial function, cytoskeletal damage, and surface expression of phosphatidyl serine. In addition, consumption of nutrients by platelets and contaminating leucocytes and accumulation of metabolic end products such as cellular debris, activated clotting factors, proteolytic enzymes, and cytokines in plasma also adversely affect the functional integrity of platelets.[8]

A battery of in vivo and in vitro tests have been adopted to assess PSL. Corrected count increment after platelet transfusion and bleeding time studies are commonly used for evaluating product efficacy and improved hemostasis, respectively. However, these do not reflect the true picture due to uncontrollable patient variables. Another alternative approach to measure the efficacy of product transfused is to measure the in vivo recovery and survival of fresh or stored radiolabelled or biotinylated platelets in healthy volunteers.[9] However, these in vivo studies are expensive, time consuming, and complex to perform.

Biochemical tests like decrease in pH, pO₂, LDH accumulation, glucose consumption, and ATP depletion are employed to assess platelet viability. Aging has also been quantified by in vitro tests measuring changes in morphology of platelets. Swirling phenomenon, decrease in MPV, reporting of the percentage of discoid forms, and Kunicki morphological score have been used for measuring the alterations in the discoid morphology. Extent of shape change in response to an agonist and increase in hypotonic shock response provide more objective measure of morphological changes.

Platelet activation markers such as release of specific granular contents into plasma (β thromboglobulin, platelet factor 4, and RANTES), changes in GP expression on platelet surface (GPIb, GPIIb, and GPIIIa), expression of sequestered granular membrane proteins (P-selectin - CD62P, CD63, and CD40L), and phosphatidyl serine (determined by Annexin V binding) on surface occurring during processing and storage have been reported.[9] Tormey and Stack observed diminution of secretory capacity of platelets during processing and storage using cytokine (RANTES) release assay.[6] Wenzel et al., used soluble CD40 ligand (sCD40L) to study the effect of storage on platelet viability by monitoring sCD40L levels in storage medium. They noticed an increase in sCD40L concentration and decrease in platelet release capacity with an increase in duration of storage.[7]

Jain et al., used these in vitro tests to observe correlation of morphological changes with platelet activation markers in platelet concentrates (PCs) prepared either from whole blood using platelet-rich plasma (PRP) method or buffy coat reduced method (BC) and apheresis platelets (AP-PC). Morphology score (MS), decrease in pH, glucose levels, lactate concentrations, soluble P-selectin (sP-selectin) levels were determined in PRP-PCs, BC-PCs and AP-PCs from day of collection to the last day of storage. A negative correlation was observed between MS and sP-selectin and a positive correlation was found between MS and pH. AP-PCs were found to be superior to BC-PC and PRP-PC with respect to quality control parameters, morphological changes, and platelet activation markers.[10]

Though in vitro tests are more economical and rapid, but none of these tests can accurately predict in vivo recovery of transfused platelets and correlation has been documented only in extreme values.[11] Moreover, majority of these tests are usually applied for research purposes and only a few tests like pH, platelet volume, platelet count, and leucocyte content have been used for daily quality control monitoring.[8]

Efforts till date at optimizing and improving the viability of stored platelets have largely focused on maintaining pH by buffering lactate production and enhancing gaseous exchange. Gaseous exchange in turn is dependent upon platelet content, gas diffusion through the plastic container, and agitation. Improved second generation containers currently in use for storing platelets (manufactured from polyolefin or polyvinyl chloride (PVC) plasticized with compounds such as triethylhexyltrimellitate and butyryltrihexyl citrate) have twice the oxygen permeability compared with first generation bags (PVC with diethylhexyl phthalate).

Growing use of leucoreduced PCs has further contributed towards enhancing platelet safety as contaminating leukocytes not only compete with platelets for nutrients but the cytokines (IL-6, TNF-α, and IL-β) generated following their fragmentation and activation have also been associated with various transfusion reactions such as febrile nonhemolytic transfusion reactions, transfusion-related acute lung injury (TRALI).[12] Leucoreduction also decreases alloimmunization and transmission of leucotropic viruses. Kaur et al., studied the levels of cytokines generated during storage and their correlation with leucocyte content. Significant rise in cytokine levels were observed in non-leucofiltered group compared with leucofiltered group.[13]

Development of synthetic storage solutions also known as PAS to store platelets is a step further in ameliorating PSL and improving platelet quality. Removal of plasma results in removal of enzymes reducing their proteolytic effect on platelet membrane. In addition, the use of PAS minimizes the adverse effects mediated by plasma like allergic reactions and TRALI, allows the use of photochemical pathogen reduction techniques, and potential improvement in platelet storage by engineered manipulation of storage medium.[14] Reduction in adverse reactions has been reported when comparing PAS preserved platelets to plasma suspended units.[15] Platelet morphology and function was found to be better preserved in additive solution on day seven as compared to platelets stored without additive solution by Chandra et al.[16]

Platelet demand has increased over the past decade as they are not only being used to control or prevent bleeding but also being increasingly used as a source of growth factors in tissue repair, wound healing, and skin rejuvenation. Studies have shown promising results of use of topical platelet therapy in various clinical conditions.[17,18,19,20] Therefore, retention of platelet functionality as much as possible is of prime importance as this may help in extension of shelf-life, and thus better inventory management.