Current methods of assessing platelet function: relevance to transfusion medicine (2005)

There are three areas in transfusion medicine where the assessment of platelet function may be informative: To identify platelet dysfunction in donors of platelet components. To assess whether changing processing or storage methodologies has an impact on platelet function in platelet concentrates (PC). To determine which patients require platelet transfusions and whether these platelet transfusions are effective by assessing platelet function in the recipient. Traditionally, platelet function tests are time-consuming to perform, difficult to quality control, lack sensitivity and usually require specialist laboratory support. However, during the last 10 years, significant developments have occurred in this field. Tests that provide more information on global haemostasis, rather than a single facet of platelet function, are now available, and some tests can be applied in a near-patient manner, rather than by specialized laboratories, so that results can be used to tailor therapy. We do not intend this review to be a ‘shopping list’ of tests that blood services should apply to PC, but aim to review methods for assessing platelet function that we believe might be relevant to transfusion and whether they have been applied in this setting. Platelet enumeration is reviewed elsewhere [1]. Overviews of normal platelet function have been published previously [2, 3]. Platelets are characteristically small and discoid in shape with dimensions of ≈ 2·0–4·0 × 0·5 microns and a mean volume of 7–11 fl. They are the second most numerous corpuscle in the blood, normally circulating at levels of 150–400 × 109/l. Platelets are anucleated cells derived from bone marrow megakaryocytes that contain cytoplasmic organelles, cytoskeletal components and platelet-specific granules. They normally circulate in the bloodstream in a quiescent state for a maximum of 10 days, but are primed to undergo ‘explosive’ activation following damage to the vessel wall. This leads to the rapid formation of a vascular plug to occlude the site of damage. Platelets are therefore enriched in signalling proteins and surface receptors that enable them to efficiently respond to vessel wall injury. Their shape and small size allows them to be forced along the edge of the vessel wall, placing them in the optimum position of maximal shear rate to constantly monitor vascular integrity. The normal vascular endothelium produces potent platelet inhibitors, such as nitric oxide, prostacyclin and a natural ADPase (CD39). However, once vessel wall integrity is compromised and subendothelial components [e.g. collagen, von Willebrand factor (VWF), fibronectin and laminin] become exposed, platelets undergo a highly regulated set of functional responses, including adhesion, spreading, release reactions, aggregation, induction of procoagulant activity, microparticle formation and clot retraction (Fig. 1). Platelet-subendothelial and platelet–platelet interactions. Reproduced with permission from S. Watson and P. Harrison P: The vascular function of platelets; in Hoffbrand V, Tuddenham E, Catovsky D (eds): Postgraduate Haematology, edn 5. Oxford, Blackwell Publications, 2004:817 (in press). Under conditions of high shear, as found in the arterial circulation, the initial platelet–collagen interactions are exclusively mediated via VWF present in plasma bridging between collagen and the glycoprotein (Gp)Ib/IX/V complex on the platelet surface. This facilitates initial adhesion or tethering, enabling platelets to slow down and roll along the damaged area of the vessel wall. Subsequent activation and firm adhesion is then mediated by other receptor–ligand interactions, e.g. direct binding of collagen to its platelet receptors GpIa/IIa and GpVI. However, at lower rates of shear found in the venous circulation, initial adhesion can also occur directly to subendothelial matrix proteins such as collagen and fibrinogen. At all shear conditions, adhesion is strengthened considerably through activation of platelet surface integrins, e.g. GpIa/IIa and GpIIb/IIIa, leading to an increase in affinity for their ligands and static adhesion. Adhesion also results in platelet activation via various signal transduction pathways and the elevation of cytoplasmic calcium levels. This causes cytoskeletal changes that mediate shape change, pseudopod formation and platelet spreading. Simultaneous release of granule components (e.g. serotonin, ADP, VWF) and thromboxane results in further recruitment, activation and aggregation of other platelets near to the growing haemostatic plug. Aggregation is mediated either by fibrinogen and/or VWF (under high shear) bridging between activated GpIIb/IIIa receptors on adjacent platelets. Platelet activation also results in the translocation of internal negatively charged phospholipids onto the outer platelet membranes, which provides binding sites for several coagulation complexes. The resultant burst of thrombin generation activates and recruits more platelets into the vicinity, but also converts soluble fibrinogen into insoluble fibrin, which is cross-linked by factor XIII, to stabilize the otherwise fragile haemostatic plug. When there is a defect in any one or more of these functions and/or in platelet number, haemostasis often becomes impaired and there is an increased risk of bleeding. The relationship between platelet count and risk of bleeding is complex. The ‘threshold’ platelet count at which platelet transfusion is required depends on the clinical status of the patient [4]. PCs are transfused to treat or prevent haemorrhage in patients with thrombocytopenia or platelet defects. These two scenarios may place different demands upon the quality and functionality of PC. For prophylaxis, platelets must be able to circulate and function as normally as possible and for as long as possible. But in patients who are haemorrhagic, an immediate haemostatic effect is desired and the potential circulating half-life might be less important. In transfusion the main application of in vitro tests has been to determine platelet function in PC. In PC, platelets are removed from the physiological environment in which they normally circulate and function: the majority of red cells and white cells are removed and, more importantly, there is no endothelium to maintain platelet quiescence. The concentration of PC (1000–1500 × 109/l) also far exceeds that of normal whole blood (150–400 × 109/l), on which many of these tests have been optimized. It may also be important to dilute control and test PC in the same media, as this is known to influence results when PC are stored in platelet additive solutions [5]. Finally, during platelet storage the ageing of the PC supernatant may also have an impact on a variety of plasma proteins and factors that are important for normal platelet homeostasis. The major disadvantage of in vitro tests of platelet function is that we do not know how the results in PC translate to platelet function in vivo following transfusion. For some tests there may be a relationship with recovery [6-10], but this has been shown mainly with PC stored in plasma; the same may not be true for PC stored in additive solutions. For many of the tests listed below, studies correlating results in vitro with platelet recovery do not exist, and indeed some have not yet been applied to PC at all. Moreover, no test has been adequately shown to correlate with the haemostatic function of platelets following transfusion in humans, partly because gold standards for assessing the clinical function of PC do not exist. The clinical utility of many of these tests (summarized in Table 1) is therefore unproven for use with PC. Nonetheless, in vitro data does provide a level of reassurance prior to the clinical use of the component, and severe lack of functionality in vitro would certainly be a major cause for concern and merit further clinical studies prior to routine production. We also need to differentiate between tests that are useful in validation studies and those that are beneficial as an ongoing check of PC quality; the majority below belong to the former category. The extent of validation is generally based upon the magnitude of the processing change and its probable impact. Small changes might require in vitro data only, whereas major changes would usually require clinical studies. The hierarchy of studies performed is usually: (1) in vitro tests; (2) recovery and survival in normal volunteers; (3) count increment studies in thrombocytopenic patients; and (4) an assessment of haemostasis (e.g. quantifying bleeding) in thrombocytopenic patients. On activation, platelets undergo a shape change, become spherical and start to project pseudopodia. When exposed to a light source, discoid platelets reflect the light and have a ‘shimmering’ or ‘swirling’ effect that disappears when platelets lose their discoid shape. Swirling is more reproducible than morphology scores [11], but the technique lacks sensitivity because assessment of a grade other than ‘yes’ or ‘no’ is subjective. PC that fail to swirl have pH values below 6·4 or above 7·6 in most, if not all, cases [12]. Thus, measurement of pH may provide similar information, but swirling is non-invasive and can be performed on all PC as a preissue check, as currently carried out in some countries. Deviation from normal discoid platelet morphology can be assessed by oil-phase microscopy and a scoring system [13]; however, the grading may not be reproducible between observers and it is a very time-consuming procedure. The extent of shape change (ESC) test uses an aggregometer to measure the shape change induced by ADP in the presence of EDTA to prevent platelets aggregating. ESC correlates well with morphology scores [14]. Although transformation from a disc to a sphere may be associated with an increase in the mean platelet volume (MPV), this in itself is not a reliable indicator, as microparticles released from activated platelets may cause a decrease in MPV [15]. There is a strong relationship (r = 0·71) between ESC and recovery in vivo, with a recovery of < 50% corresponding to ESC levels of < 60% of day-1 values [6]. However, in an changes in platelet morphology no with of haemostatic function in Aggregation is usually assessed by plasma at in a between a light and a Aggregation is by such as collagen, ADP, or or in platelets start to there is an increase in light and results are as the rate of aggregation and maximal to different platelet can be and from and aggregation studies therefore an important Platelet aggregation in whole blood can also be by based on and by the decrease in platelets that in to The can be or by a disadvantage of aggregation is that it tests platelets shear and in to single conditions that are not of haemostasis in Platelet aggregation in PC does not to platelet PC stored at maintain aggregation than those stored at lower recovery and studies have shown to single during PC However, this effect is when of are and the of stored platelets to respond to ADP or collagen may following transfusion the of platelets stored for is increased if in plasma rather than in plasma from the PC which in be by the of The platelet function by to measure platelet adhesion, activation and aggregation conditions of high shear The has been by the is applied to a a with either or and which has a into Under high shear rates of blood with the causes platelets to and occlude the The to occlude the or is the that is in an to the bleeding The maximal that can be is The technique is and but to a of including platelet number, blood and plasma VWF levels. a normal has a high it is to become more as a test for platelet and results may The test is more than the bleeding in von and various other platelet and is also to various The has been used to that a significant of donors or donors have platelet dysfunction at the of This to be partly because of an of or other inhibitors, as well as and is in the same donors Moreover, in the to decrease platelet function, as assessed by the These studies that for donors who have do not prevent and that such as the might be a useful check, for However, it is not yet whether PC from donors with results are less In a small of transfusion of PC from donors with not correlate with increased blood and platelets from donors bleeding in vivo, the effect and bleeding that of circulating platelets have not been PC from donors who have to be These studies are as has been to the of to platelet function in PC. the is for use with whole blood and high shear conditions, from PC be assessed This can be by PC into whole blood by whole blood or red cells and This has been used to that increase during platelet a of platelet function However, can become the of platelet these can be by to the of the of blood and PC the of when the a rapid by the with normal platelet The by and uses a blood usually or of blood is through the the This into a system that does not require the use of blood The of two one of which a with proteins (e.g. or are a set of by grading single platelets or or normally however, microscopy and are used to this to be by the between the the or the of the The main of is that platelet and formation can be conditions of These are not used in clinical but have found application in the of platelet function and the of various studies that adhesion to collagen, matrix or blood is by storage of PC, whereas a decrease in platelet of to 50% by tests that have been used to or platelet and are the and and the Platelet the but these have not been applied to PC. The platelet aggregation based on light whereas the the adhesion of platelets to matrix or whole blood exposed to high shear which is by a in the The has now been as an the rapid of of platelets small of There are a of and that can be used to platelets by of their application are shown in Table can be used to assess changes in platelet that occur during storage of PC, including platelet glycoprotein (e.g. the generation of platelet activation the of negatively charged and the formation of studies platelet that are important in platelet adhesion and aggregation in PC during storage have on and When platelets become GpIIb/IIIa a change it to fibrinogen and are now that on GpIIb/IIIa which become exposed during this rather than in the fibrinogen to the platelet or the of fibrinogen to to receptors in to can be The may be more informative: of GpIIb/IIIa does not to change during the storage of PC, whereas there is a in fibrinogen binding to platelets in to thrombin The of can be by various in a to VWF and There is a strong between the of platelets for and in vivo the of in platelet has been in When platelets are stored in the there is of on the platelet which results in by receptors on and rapid from the The of on the surface of platelets is used as a of platelet yet its clinical is is in platelet but is to the surface when platelets become It has been that increased of may be for an increased rate of of platelets from the However, in platelets with thrombin to cause of granule have a survival in the that is not different to that of platelets platelets are not able to but to be functional once with generation of and a has been shown to be in platelet data from a thrombocytopenic that changes in may be in the rapid of platelets from the circulation, whereas changes in are in of platelet of is that it provides information on the of cells a and the extent to which it is the is to is that proteins can be from the surface by the of various and this not be For and can be from the surface of the platelet and it has been that the measurement of soluble in the supernatant may be a of platelet activation Moreover, platelets are generally assessed in their The of and GpIIb/IIIa well as fibrinogen to in to physiological but this is impaired during platelet storage also that if this is induced by it can be or by the of plasma and storage The of platelets to rather than glycoprotein might be a of in vivo but this to be The of phospholipids on the platelet and the generation of microparticles following activation, results in a increase in the rate at which factor can to thrombin and provides a surface on which factor can be activated by of thrombin of on the platelet surface can be by the binding of In the platelet has been assessed of the where rather than provide generation and in plasma or whole blood can now be or coagulation factors can be to platelets and thrombin generation assessed by a on platelet during storage of PC is some studies an increase and a because of the methods used Platelets contain that are for platelet 60% of ADP and are stored The is present in to or in the There are two major pathways in and via the for are and rather than The majority of in platelets is derived from which produces as an that can out of the storage However, the of is and that The is in plasma by which is when levels ≈ which there is a rapid decrease in When PC are stored in pH values below are associated with in vivo recovery For this pH is used as a routine test by most blood The relationship between high pH and is less studies have shown a of recovery in vivo at pH values of whereas that PC with a pH of to 7·6 do not a of in vitro function or in vivo of is the which is based on the of platelets to and the rapid that when in a and can be by an There is a between (r = or levels and in vivo [6]. in vivo recovery of < 50% to to an or an level of < of platelets. There is a (r = between platelet and survival [6]. or has been well cells and has been to be an important during platelet formation from megakaryocytes platelets may a specialized of that are not and removed by further ageing in the or by in vitro of The for of platelets from the but have also been platelets. platelets normally potential and a However, upon in vitro storage of a of changes have been including platelet cytoplasmic plasma or microparticle of of potential and of these important changes can be by has been in stored but there are data to the and the at which it is in most studies this or the of platelets in this area is very but may to important of the platelet storage and platelet These tests do not measure platelet function in but rather in the of clot formation and they are also on plasma coagulation and or whole blood can be The system has been as a based on a by and three platelet clot and thrombin generation small of whole blood is between two and is by various the clot platelets to the and a is to the of which is by a The can be into by of a the The does not of platelet and is in platelets stored at of PC with to prevent of the the of PC to of that of PC, yet has effect on blood as in a This that this in vitro does not to haemostasis following transfusion. The more than blood is in a in which a is that is to a or The in In blood the is but as the blood the of the is to the whole blood or plasma can be with or of the factor or factor The is rapid to can be in a near-patient and it provides various data to clot formation and the the clot to the rate at which the maximal of the and the extent and rate of is an of the where the is and the and that clot is storage of PC for of platelet recovery and survival following transfusion to normal is by many in the to be the assessment of PC quality and is usually performed by an of PC and to the 10 and recovery and survival can be There are a of to such as the is and must be to red can also become of the platelet it is difficult to on PC, as they are from more than one and, some are not to out either or studies. no has been as to values for platelet recovery and survival following but a has been which that the mean recovery should be of platelets and survival 50% of platelets However, the of the and and other are yet to be and survival can also be assessed in thrombocytopenic patients more count increment studies are performed in this setting. either the data provides information on the functionality of the it is a for platelets to be able to circulate in to At platelet of < × there is an relationship between platelet count and the bleeding in some patient and in bleeding to be the test for platelet function, but is now as it is reproducible between and not for tests the same bleeding as an measure of platelet transfusion is because it is but there are a of in this are the site and of and whether data are in a or in these factors can to significant between studies. Although most studies use the grade bleeding or an of there is a lack of a grading system and which are the most relevant of patients may need to be at bleeding where occur assessment of bleeding has been used in studies platelet the for platelet and more for platelets have been to assess platelet function in PC, the most used the platelets transfused into are by the this must be are thrombocytopenic by the use of or other and survival can be in that are not by platelet The main and of this technique is an assessment of haemostasis by of bleeding from an induced injury. This is usually a in the but other have been such as injury. have been useful in assessing platelet which are difficult to assess in They have also been applied to the storage of platelets of platelets and storage of either or studies have also used the or to the of PC transfusion In many patients have that are to measure as a of either thrombocytopenia and/or platelet However, in some patients there is a of the following transfusion of PC. This is in than platelets and to a relationship to in the control of bleeding the also that some patients normal results they that they may not have required a platelet transfusion. studies that the do not correlate very well with platelet count some patients to a count but a in that platelet function further has shown that there is a between results following transfusion and those if a from the patient is with the same PC that transfused Platelet adhesion to is in platelets stored at with a of the bleeding following transfusion of either and with PC. of significant in of assessing platelet function in transfusion we know we platelets and function we need them to We have to whether platelet transfusions are or whether in patients who are bleeding might be more studies in this area may of the functional of an PC should PC transfused in a some that are of as such as platelet activation and might be highly and which recovery or but are may increase in as platelets and platelet do we in vitro tests to survival or haemostatic have shown that tests which one might not the it is to that one test provide all the in platelet function in PC are and which are The in which we test PC may be to the results and we may need to more in this is the and clinical of platelet dysfunction in blood we which patients require platelet transfusions and assess whether they are effective by or of the tests of platelet function may to these but not many have been applied in a transfusion and some are difficult to to PC. They require validation relevant in and clinical studies.