The platelet storage lesion

The Platelet Storage Lesion Jerard Seghatchian and Pranee Krailadsiri

URING THE PAST 2 decades, the demand for platelet concentrates (PC) has been steadily increasing because of more intense treatment schedules as well as widening indications for the use of chemotherapy. This has. placed considerable pressure on the logistics of supply. The emphasis on reducing donor exposure, caused by the possibility of transfusion associated alloimmunisation and leucocyte borne infection, t has led to an increase in the use of apheresis and leucoreduced PC. In re@onse to these requirements, measures have been taken that are aimed at maximum resource use, for example: by splitting a high platelet yield unit, collected by a cell separator, into two therapeutic doses (-->240 • 109/dose); development of new containers and storage media to permit prolonging the length of storage period; and the development of new generation of leukocyte filters as welLas modem apheresis procedures to achieve high purity leukoreducedproducts (~1 • 106/unit). In parallel to the above development, the bottom and top (BAT) bag system, 2 combined with semiautomated blood component separation procedures, have been used to optimize platelet recovery from whole blood and to standardize the yield. This has enabled the consistent production of PC with an average dose of 300 X 109 platelets/unit and leucocyte content 0.05 X 109/unit from a pool of four ABO compatible buffy coats. Storage in synthetic media has been proposed 3 with the practical advantages of reducing the rapid accumulation of lactate and the removal of some plasma components, cell-released enzymes, and soluble antigens including HLA; therefore improving the viability of platelets as well as reducing potential transfusion reactionsJ -6 Despite these continual process improvements and major advances in establishing optimized storage conditions, the platelet storage lesion (PSL) as manifested by the loss. of function and changes in morphology associated with vacuolisation and plate-

D

From the National Blood Service2-London and the South East, Colindale Avenue, London, UK. Address reprint requests to Dr JMJ Seghatchian, NBS-London and the South East, Colindale Ave, London NW9 5BG, UK. Copyright 9 1997 by W..B.Saunders Company 0887- 7963/97/1102-000553.00/0 130

let activation followed by microvesiculation or fragmentation continue to occur.7-I~This, at least on a theoretical basis, could be associated with a decrease in posttransfusion recovery and the length of time platelets remain in the circulation. 6 Nevertheless, no clear cause-effect relationship between decreased function in vitro and the decrease platelet viability in vivo has yet been established. E~en if a good correlation between the results of various laboratory measurements and clinical outcome are expected, there are situations in which such correlation may not exist. Thus, no single laboratory test alone reflects the eventual hemostatic function of platelets in vivo. The projected increase in demand for high quality PC therefore makes it necessary to unravel the mystery of the platelet storage lesion (PSL). Such knowledge could lead to the improved preservation of platelets, therefore improving clinical outcome. This overview highlights some of the key features of the PSL, focusing on influencing factors, the current available markers of the PSL and of platelet transfusion reactions. Emphasis is placed on practical tests for monitoring for the PSL, ie, shape changes, and tests reflecting functional integrity, activation state and presence of microvesiculation. The role played by some biological response modifiers (BRM) such as biologically active cytokines in the development of-PSL and the possible toxic effects of bacterial growth, in some stored PC, on transfusion reactions also are reviewed briefly. DEFINITION

There is as yet no generally agreed definition of the PSL as the mechanism of the lesion is not fully understood, v,8 The definition used by the authors is that the PSL encompasses all the (deleterious) changes in platelet morphology, structure and impaired platelet function from the moment of blood collection from the donor to the administration of a PC to a patient. Although this definition lacks precision, as it does not describe-the process(es) responsible for the observed changes, it is a convenient one because many of the changes that take place during storage Fan be identified and quantified by a variety of in vitro tests. This definition, however, does not include three important aspects of metabolic changes which take place during Transfusion Medicine Reviews, Vol 11, No 2 (Aprili, 1997: pp 130-144

THE PLATELET STORAGE LESION

storage and which should not be neglected. Firstly, there is a progressive change in the activity state and the concentration of many plasma constituents. These arise either as the result of the metabolic activity of platelets (or leukocytes present in PC) or represent the continuation of reactions occurring in the plasma at the time of blood collection. The presence of metabolic end-products such as; activated clotting factors, cellular debris and proteolytic enzymes, is undesirable as it may affect profoundly the functional integrity of the platelets in a PC, and may contribute to transfusion reactions in recipients. 9-1~Secondly, this definition does not provide information on the potential effect of some metabolites either on in vivo recovery, survival and hemostatic function of transfused platelets; or on the in vivo platelet production, ie, through bioregulatory role of generated cytokines. 11 Thirdly, at present it is not clear which changes remain irreversible and to what degree transfused platelets can be rejuvenated in vivo.l~ PLATELET SHAPE CHANGES

In clinical practice platelet morphology is the best indicator of the ability of stored platelets to remain in the circulation. Circulating platelets collected in a citrate solution retain their discoid shape although they may exhibit a number o f tendrils seen in early stages of activation. 12,13This discoid appearance is gradually lost within a few days of storage at 22~ and at the same time there is an increasing number of microvesicles that arise from the separation of tendrils and fragmentation of the cellular membrane. 14 After 5 to 7 idays of storage at 22~ almost all platelets in PC that are below the acceptable pH (<6.4) for storage, are spherical or in fragmented forms. 1~ Whether the young, large, and more dense platelets have a longer shelf life and retain their functional integrity better than small and less dense platelets has yet to be shown: Nevertheless, both platelet size and aggregation response decrease rapidly during storage, though experimentally aged platelets can be rejuvenated by resuspending them in fresh plasma. 10 This, on a theoretical basis, may be related to the in vivo recovery of stored PC, as shown schematically in Figure 1. Thus platelet shape change, as manifested by discoid/spheric conversion and associated with cemralisation of granules, still remains the most practical marker of the overall PSL. t0 The development of a simple and practical method for

131

1

2

3

4

5

1

2

Fig 1. Schematic representation of in vitro changes in platelet function over 5-day storage; and the expected in vivo transfusion response. (A) Gradual decrease in platelet M P V ( ~=2 fL) caused by discoid/spheric conversion and microvesiculation, by the end of shelf life. (B) Progressive decrease in platelet aggregation response t o ~ 2 5 % , depending upon metabolic activity of the cellular content. (C) and (D) Exponential rise in the levels of biologically active cytokines (C) and C3a (D) to clinical significant levels at day 3, depending ,the concentration of residual leucocytes in PC and depending on the activation states of the plasma components respectively. (E) and (F) The expected and the observed clinical response as 30% of platelets go immediately into the spleen and may return to the circulation after 6 hours.

the estimation of platelet shape change has been the focus of attention of many investigators for many years. A practical approach for the evaluation of platelet shape changes based on the size distribution by light scattering is described later in this review. PLATELET ACTIVATION

Platelets undergo activation in response to a variety of stimuli as well as exposure to foreign surfaces and mechanical trauma.l~ This is associated with the formation of microaggregates of two to twenty platelets, an event that requires subtle conformational changes in the platelet glycoprotein IIb/IIla complex, making them available to fibrinogen and other adhesive proteins such 'as von Willebrand Factor (vWF). Upon binding of the GPIIb/IIIa complex to its ligands, further conformational changes occur leading to the changes in platelet cytoskeleton and the clustering of the receptors in activated platelets.16,17 A resting platelet contains 50,000 copies of GPIIb/IIIa on its membrane surface, with 30,000 in the internal membrane pool. 17 The number of this receptor expressed on the platelet surface upon activation varies, depending on the nature of the stimuli.

SEGHATCHIAN AND KRAILADSIRI

132

Thrombin is considered a strong agonist and ADP a weak agonist, leading initially to reversible aggregation. In developing the methods for assessing PSL, it is, therefore, relevant to characterize the nature of reversibility of platelet aggregation as previously described. 18,19 The complex set of shape changes, activation, adhesion, and aggregation, which take place in platelets during the storage of PC, is similar to, but not identical with the activation of fresh platelets under laboratory conditions. Firstly, the storage lesion of stored platelets occurs over several days in which metabolic exhaustion after handling may contribute to the PSL, whereas activation of fresh platelets and their transformation from resting to the exhausted form occurs in seconds and is completed in minutes. 12,13,17 Secondly, platelets in 5-days stored PC, which are unable to aggregate when stimulated with low concentration ADR still possess the capability to aggregate after incubation in fresh plasma. 1~ Thirdly, stored platelets, which exhibit the high level of P-selectin on the membrane, have reasonable recovery and almost normal survival in vivo. 6,20,21 In contrast activated fresh platelets, which have passed the stage of reversible aggregation are committed to complete the secretion reaction, which leads to the demise of the cells. FACTORS INFLUENCING THE PLATELET STORAGE LESION

Several factors may influence the rate of production of the platelet storage lesion and platelet activation to variable degrees, as summarized in Table 1.

Effect of Collection Techniques Platelet concentrates can be harvested from whole blood as platelet-rich plasma (PRP), as buffy coat (BC), or by plateletpheresis as shown in Figure 2. In the PRP technique, the intermediate PRP product is highly heterogeneous and contains high levels of leukocytes and red cells. In practice it is extremely difficult to standardize this procedure as depending upon the biological differences in donors' hematocrit (Hct), variable gravity forces are exerted on the cellular content of blood during the first spin. Therefore, even in a validated system 30% to 40% of PRP preparations are heavily contaminated with other cells. Moreover, the high gravity force used in the second spin for "pellet-

Table 1. Factors Influencing the Rate of Development of the Platelet Storage Lesion Collection Techniques 9 Composition of anticoagulant/preservative solution 9 Blood flow rate 9 Ratio of anticoagulant 9 Centrifugation force (acceleration/deceleration times) 9 Resting period before resuspension Storage Conditions 9 Temperature and length of storage of whole blood before and during processing and storage 9 High cellular content (PLT, WBC, RBC) 9 Volume and composition of suspension media 9 Final plasma concentration in the storage media 9 Type of agitation Characteristics of Storage Containers 9 Plastic bag composition 9 Pack size and thickness of plastic 9 Gas transfer properties of plastic 9 Thickness of wall of container Treatment after Collection 9 Extent of leukodepletion 9 Extent of plasma removal 9 UV-B irradiation 9 3,-irradiation 9 Cryopreservation 9 Lyophilization

ing" platelets on a plastic surface, activates platelets and often leads to irreversible aggregates. 19,~~ Whenever platelets are brought together in close contact, there will be irreversible changes which may compromise platelet function both in vitro and in vivo. zl,22 In the BC technique using the bottom and top (BAT) blood bag system, 2 the volume of BC is standardized to recover 80% or more platelets and leukocytes. Moreover, in this protocol the platelets are pelleted on a cushion of red cells rather than the plastic bag producing less platelet activation. In addition as the second centrifugation step is carried out on a pool of 4 to 5 BCs, with a constant Hct, a more homogeneous gravity force is exerted on the platelets. Therefore, platelets derived from pooled .BCs ~ e less activated and more homogeneous, containing less aggregates than PRP-PC. Moreover, this process lends itself to fine tuning and standardization, therefore with a validated protocol it is p0ssible to obtain a reproducible dose (containifig~300 • 109 PLT/pack) in which a high proportion (30%) contains less than 5 • 10 6 leukocytes witfiout leukocyte filffation. 22At least four variants of BC processing before pooling exist currently, the so called wet (BCs of -=-120 mL), dry (buffy coat of 65 ~ ) short and long hold (single BC, stored for

THE PLATELET STORAGE LESION

133

1. The PRP protocol

2. The buffy coat protocol

Unit of whole blood in CPDA-1

1st centrifugation I

Unit of whole blood in CPD

1st centrifugation J

2,800rpm x 12min

I

red cells

3. Plateletpheresis using ACPD

3,000rpm x 12min

I

platelet-rich plasma

buffy coat

red cells

plasma continous or discontinuous protocols

2ndcentrifugation 1 3,000rpmx 7min 2nO ] 2,a00rpmx 3m,n

+ plasma Fig 2.

platelet concentrate

platelet concentrate

buffy coat residue

platelet concentrate

Preparation of platelet concentrates from a unit of whole blood either by two centrifugation steps or plateletpheresis.

< 6 hours and up to 24 hours respectively). Although PC derived from these variants are comparable on the basis of their cellular content, those from the short hold BCs may contain larger platelet aggregates than those from long hold BCs, whereas the long hold BC-PC are often associated with elevated levels of biologically active cytokines in PC at day one 1~ (Table 2). This may also influence the relatively poor long term stability during storage of BC-PC as compared with PRP-PC or standard apheresis-PC (Fig 3). In apheresis techniques, blood cells are subjected to various degrees of collection/separation shear stress depending upon the design and/or whether continuous or discontinuous flow is used. Moreover the pelleting on the surface of plastic bag (ie,

as in Fenwall CS3000) and vigorous agitation of PC for resuspension can grossly influence stability during storage~ The flow rate and the gravity force are usually preprogrammed by the manufacturers for optimal performance and depending upon the type of apheresis machine used, products derived from standard apheresis machines are relatively heterogeneous with respect to recovery of the various subpopulation of platelets and leukocytes, as well as the quantity and nature of the biologically active cytokines (Table 2); 10'11'22

Effect of Platelet Storage Containers Many years ago, the same type of plastic bag was used for both the collection of blood and for platelet storage. Today several types of containers

Table 2. The Average Cellular Content and Cytokine Levels in 4 Types of Platelet Concentrates (PC) at Day 1 and at the End of Shelf-life Parameters Days WBC (• 109/L) PLT (x 109/L) IL 1 (pg/ml) IL 6 (pg/ml) IL 8 (pg/ml) TGF~ (pg/rnl)

PRP

BC-PC

Hemonetica

Cobe-LRS

1

5/6

1

5/6

1

5/6

1

5/6

0.9 1538 ND 140 100 11050

0.7 1480 24 2395 32438 21000

0.16 933 6 73 489 2900

0.16 933 ND 78 440 5850

0.55 1322 ND <15 51 23666

0.44 1093 ND <15 228 72000

ND 942 ND <15 170 2670

ND 906 ND <15 180 27900

Note: The drop in platelet count during storage, in particular in packs containing ->1300 PLT • 109/L indicates cellular fragmentation and microv~siculation. PRP-PC showed the highest concentration of leucocyte and inflammatory cytokines whereas Hemonetics apheresis PC showed the highest concentration of platelet derived TGFI3. The long hold BC-PC showed the highest IL-8 level at day 1 which then remained unchanged or slightly decreased during storage. Abbreviations: ND, not detectable.

134

SEGHATCHIAN AND KRAILADSIRI

Moreover, this citrate-based plasticizer can be metabolised by platelets through the tricarboxylic acid cycle into physiological useful compounds. 15 To what degree the different types of containers influence the production of the PSL remains an issue of interest.

PRP-PC

25I 2.0 1.5 10

0.5

=, | 6.00

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. 8o0

6 0 -0.5

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BUFFY COAT-PC

Effect of Storage Temperature pH

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6.~o

8.oo

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HAEMONETIC-PC zs,

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C

pH

Fig3. The relationship between dMPV v pH for three types of platelet concentrates produced at North London Blood Center. (A) The results of random donor PRP-derived platelet concentrates, (B) Buffy coat poor platelet concentrates; and (C) Apheresis platelet concentrates. The fresh units (filled squares) have high pH and high dMPV whereas the aged units (empty squares) have lower pH and sometimes negative dMPV. A large number of PC-derived from either PRP or buffy coat methods, in contrast to apheresis, show unacceptable pH or dMPV. The fall in dMPV is more rapid in pooled buffy coat platolet concentrates.

with different shapes, sizes, and thickness are designed for PC storage. The first generation of platelet containers consisted of relatively thick polyvinyl chloride (PVC) and 2-diethylhexylphthalate (DEHP) as a plasticizer. This type of container did not allow adequate gas exchange to enable platelet storage beyond 3 days and was associated with the development of a toxic compound monoethylhexylphthalate (MEHP) derived from the releasable plasticizer. 23,24 This metabolite also impaired the aggregation response of platelet by inhibiting phospholipase A2 and produces cardiotoxic effects.23,24 However, most new generation containers use triethylhexyltrimellitate (TEHTM) and a citrate-based plasticizer, butyryl trihexyl citrate, which seems to be less soluble in plasmaY

Although it was once a common practice to store PC at 4~ it was noted subsequently that the recovery and survival of the platelets store'd at this temperature were decreased relative to platelets that had been stored at 2 2 ~ 26 Cold-induced storage lesions include irreversible loss of discoid shape, caused by depolymerisation of microtubules and the breakdown of platelet contractile protein with loss of ATP.26 However cold stored PC are hemostatically effective with the ability to rapidly correct bleeding time, whereas there is a lag period with 22~ stored platelets, such that they may not correct the bleeding time for up to 24 hours following transfusion. 26There is now general agreement that a 22~ hold combined with agitation of the PC during storage to enable good gas exchange, in an oxygen permeable bag is essential. 25 Recently 9 the rate of loss of platelet viability during in vitro storage at 22~ was compared with that at 37~ was reported, clearly indicating that at 22~ the rate of aging is substantially reduced as compared with that seen at 37~ 27 This may be related to the lower metabolic rate during storage at 22~ In view of relatively high frequency of transfusion-associated sepsis from bacterially contaminated PC, stored for up to 5 days at 22~ the issue o f the temperature of storage remains a focus of interest. It seems that gram positive organisms reach maximum growth by day 3, which is somewhat more rapid than the growth for gram negative organisms. 2 Therefore, reducing the length of platelet storage before transfusion, with the hope of preventing bacteriologic complications and reducing the associated storage lesion, may prove beneficial.

Effect of Storage Media The storage of platelets in a nonplasma medium was first reported in the mid 1980's by Rock et al and subsequently proposed by others with some slight changes in composition, ~using a variety of different salt solutions? In principle, the removal of plasma from PC results in removal of plasma

THE PLATELET STORAGE LESION

enzymes reducing their proteolytic effect on the platelet membrane, in particular glycoproteins Ib/ IX/V and IIb/IIIa complexes that are essential for the adhesion of platelets and for their participation in the aggregation response. It has also been documented that the use of synthetic storage media with inhibitors is associated with improved viability, reduction of bacterial content, and better maintenance of other in vitro parameters. 28 Nevertheless a balance ratio between the residual plasma and suspension media is needed, where the influence of some storage containers in the increase rate of thrombin generation should not he ignored. 29

Effect of Residual Leukocytes The significance of leukocytes in PC should be looked at on the basis of four different information sets. The first set relates to the consequences of the inevitable activation or fragmentation of leukocytes during storage. On the one hand, leukocytes compete with the platelets for the nutrients in the plasma, whereas on the other hand leukocytes release enzymes (eg, elastase), vasoactive substances and biologically active cytokines, (Table 2), which may reach sufficient concentrations to cause transfusion reactions in recipients. 9-11 The second set of information relates to leukocytes whose presence leads to a number of direct adverse effects in transfused recipients. 1,3~The removal of leukocytes by filtration to produce leukoreduced PC, although useful in reducing some side effects, may cause deleterious effects on platelet storage stability. 31 Thirdly, leukocytes present in blood play an important role in the bactericidal system of the donated blood and PC within the first 22~ to 48 hours after collection. 2 It is likely that the phagocytic capacity of the granulocytes presen't is sufficient to remove the small burden of bacteria that may be present on the surface of the skin and occasionally enter the blood bag at the time of venepuncture. 2 In this respect, in line filtration or complete depletion of granulocytes during the early phase of processing, on a theoretical basis, may make PC more prone to bacterial growth. Moreover, as mentioned previously, certain types of bacteria may, enhance the development of the storage lesion and contribute to transfusion-related sepsis. 32 Finally, any additional processing, such as the transfer to a new container, the removal of residualqeukocytes by respinning or different types of leul~ocyte filters, the removal of plasma, wash-

135

ing, cryopreservation, UV-B and ~/-irradiation can influence platelet storage lesion to variable degrees.33-38 ASSESSMENT OF THE PLATELET STORAGE LESION

Platelets in PC undergo a number of changes during collection, processing, and storage which can affect their structure and function. 1~176 These changes can be quantified by a number of in vitro tests that have been primarily designed for diagnostic purposes (Table 3). However, there is no established yardstick that can be used to translate the measured loss of platelet function into an appropriate scale of PC quality. It is not possible to indicate when, within the period of storage, a viable platelet becomes nonviable. The following are some useful and practical new tests to indicate the potential presence of the PSL.

Changes in Platelet Size, Shape and Aggregation State There is a resurgence of interest in establishing whether there is a relationship between platelet size, density, and viability of stored PC. 41,42Normal platelets display a log normal size distribution pattern when analysed by an automated cell counter. 33 Using an automated cell counter based on laser optic technology (Technicon*H series) we have observed that in the presence of small aggregates (doublet/triplet), there is a right shift (high mean platelet volume, MPV) and in the presence of larger aggregates, new populations of cells appear as pseudo-erythrocytes or pseudo-leukocytes in the histograms produced. 33 In contrast, upon microvesiculation and fragmentation, there is a left shift in platelet size distribution pattern, with a distinct population of cell fragments with an MPV of less than 3.8 fL. Therefore, changes in cellular indices and the size distribution of platelets, red cells, and leukocytes are of value in assessing the dynamic shape change and the aggregation or disaggregation state that platelets undergo during storage. 19,43 Platelet shape change can be measured by a number of different methods, 41-4a including the observation of the swirling phenomenon. This procedure seems to be the simplest way to establish whether platelets have undergone shape changes, and by inference become nonviable, but this measurement is rather subjective. 44 The quantitative assessment of platelet size distribution pattern (ie

SEGHATCHIAN AND KRAILADSIRI

136 Table 3. Current Methods for the Quality Monitoring, for Process Validation and for the Development of New Products/Processes for Platelet Concentrates Measurements Reflecting Compliance with Specified Minimum Requirement 9 Plateiet concentrate volume measured by weighing. 9 Visual inspection and the determination of swirling property. 9 Glycolytic property measured by pH. 9 Cellular content (PLT, WBC, RBC). 9 Platelet size distribution measured by an automated cell counter having a good linearity range as well as sensitivity. 9 Ability to detect with precision low levels of leucocytes (ie, <5 • 106 per unit), at least on pass/fail principle. 9 Ability to differentiate pseudoleucocytes/erythrocytes (platelet aggregates) from the real cells, based on changes in cellular indices with and without addition of EDTA, Measurements Reflecting Metabolic Activity 9 Rate of pO2, pCO2, and pH change. 9 Rate of lactate production. 9 Rate of glucose consumption. 9 Reduction of ATP concentration or change in ATP/ADP ratio. 9 Presence ofthromboxane metabolites. 9 Rise in intracellular calcium. Measurements of Shape Changes or Morphology Index 9 Morphology score or percent discoid (oil-phase microscopy). 9 Hypotonic stress (osmotic reversal) reaction. 9 Extent of shape change. Measurements Reflecting Change in Platelet Aggregation 9 Spontaneous aggregation. 9 Aggregation in response to pairs of agonists. 9 Changes in vWF:Ag levels or its collagen binding activity vWF:CBA) or ristocetin induced aggregation; or vWF:Ag multimeric patterns. Measurements Reflecting Platelet Activation, Secretion and Lysis 9 Reorganisation of platelet prospholipids assessed by labelled Annexin V. 9 Expression of activation dependent markers on the platelet surface (GPIb, GPIIb/llla, P-selectin, vWF and Annexin V, etc). 9 PF 4 and 13-thromboglobulin in platelet supernatant. 9 vWF:Ag and vWF:CBA in platelet lysate and/or supernarant plasma. 9 Cellular fragmentation resulting in microvesicles in platelet supernatant measured by either procoagulant or anticoagulant properties. 9 Microvesiculation using different markers of platelet storage lesion, ie GMP-140, glycocalicin, vWF, Annexin V, Gpllb/l!la and HLA. 9 Content of lactate dehydrogenase (LDH) in platelet supernatant. Measurements Reflecting the Possibility of Transfusion Reactions 9 Bacteriological growth. 9 Cytokines (in particular biologically active forms). 9 Activated complement components; in particular C3a.

Table 3. (Cont'd) 9 Activated clotting factors. 9 The presence of low grade proteolytic enzymes or their complexes with various inhibitors. 9 The formation of neoantigens on platelets and their membraneous microvesicles. Measurements Reflecting the Presence of Circulating Young Platelets 9 Concentration of reticulated platelet. 9 Concentration ofThrombopoietin (TPO).

MPV), in particular with light scattering devices, is preferable,41,43 but it provides limited information unless the measurement is carried out twice. Firstly, after preparation of PC to assess which population of platelets are recovered; and then subsequently during a set period of storage, so that the changes in MPV caused by storage can be determined. Thus the determination of a sample at a single poirit during storage does not provide enough information on the activation or aggregation states of platelets. The method preferred by our laboratory is the measurement of difference (d) of MPV before and after the addition of 0.5 mL of a platelet sample to commercially available 4 mL dry K 2 EDTA tubes, holding for a fixed period (ie, between 15 to 60 minutes for optimal effect) before counting, and the calculation of the difference in cellular indices by the following formula.43 dMPV = MPV(+EDTA) - - MPV(citrated) dPLT = PLT(+EI)TA)-- PLT(citrated) A comparison of different types of PC with respect to the above parameters reflecting functional integrity (dMPV) versus pH is shown in Figure 3. Although all types of PC seem to-be equivalent in terms of platelet and leukocyte content, BC-PC contains the least number of aggregates as measured, by dPLT, whereas PRP-PC not only contain a large number of aggregates, but also have the smallest subpopulation of platelets possibly a relatively higher content of microvesicles, therefore less functional platelets as indicated in Table 4. Of particular relevance to the functional integrity of platelets is the response of whole blood or PC samples to the cold (4~ which leads to platelet shape changes and pseudopod projection.33 Although these changes, as measured by MPV, are initially reversible upon rewarming, the prolonged

137

THE PLATELET STORAGE LESION

Table 4, Quality Parameter of 5 Types of PC Produced at the North London Blood Center Volume (ml) PRP-PC (n = 600) Pooled BC-PC (n - 1000) Apheresis PC (PCS+) (n - 1100) Apheresis PC (MCS) (n = 1200) Apheresis PC (Spectra) (n = 300)

48 325 219 224 259

Platelets (• 68 296 280 303 365

Leucocytes (• 0.03 0.06 0.07 0.08 0,003

pH

dMPV (fl)

dPLT (xl0Eg/I)

7.35 7.16 7.30 7.19 7.31

0.56 0.70 1.39 0.95 1.37

161 62 139 274 140

Note: Platelet preparations are heterogenous in terms of the conventional quality parameters and tests reflecting functional activity (dMPV) and aggregation state (dPLT). A lower dMPV indicates loss of the platelet ability to undergo EDTA induced shape changes; a higher dPLT indicates the presence of greater numbers of aggregates in a PC,

storage (overnight) at 4~ leads to irreversible shape changes. 39,4~This is understandable as prolonged storage at cold temperatures disassemble platelet microtubules, 45resulting in platelet dysfunction and microvesiculation. 46,47 We have observed that platelets that are already in the spherical or in an activated state will not respond to cold exposure (2h) to the same degree, ie, 0.2-0.5 fL increase in MPV when stored for 5 days, as compared with 2 fL when fresh. 10,47Thus the paired sampling protocol comparing the cellular indices of the cold treated samples, (ie, 2 h exposure to 4~ with that stored at 22~ can also be used for the estimation of the PSL, platelet activation and the capability of platelets to further undergo disc/spheric conversion. 10

Spontaneous Aggregation Samples from all types of fresh PC undergo spontaneous aggregation subsequent to pre-analytical mixing (3 mL in plastic tube) and sequential counting by a Technicon cell counter. This phenomenon seems to be specific to the type of reagent used by this cell counter which fixes cells before their measurements. Spontaneous aggregation is characterized by a drop in the platelet count after a lag phase (5-15 minutes) and a concomitant increase in both MPV and pseudoleucocyte count (platelet aggregates) detected in the peroxidase channel 33 and finally a drop in MPV and the size of aggregates possibly due to surface and/or shearinduced microvesiculation. We have observed that the collection- or processing-induced generation of even trace amounts of ADP or thrombin dramatically reduces the lag phase of spontaneous aggregation, whereas'the addition of disaggregating agents, eg, apyrase or EDTA, prolongs this lag phase. Moreover/various processing steps, such as leukocyte fil~ation can also contribute to platelet injury as identified by the shortening of the lag phase and

increased aggregate size measured by the ratio of peroxidase/basophil leucocyte count. 33 In view of the high sensitivity of platelets to undergo spontaneous aggregation, we have adopted this procedure for establishing the best practice and for our in process quality assurance validation program. 18,19,33

Platelet Function Testing Current assays for assessing platelet function are relatively insensitive to define with accuracy and reliability the relatively small degree of platelet injury that occurs during processing and 5 days storage, particularly when PC are kept under t h e i r optimal storage condition. For example, serotonin uptake by platelets is unaltered during storage; the expression of platelet activation markers, GPlb and GPIIb/IIIa, remains either stable or decreased by no more than 15%; and the level of lactate dehydrogenase is increased about 10% and PF3 availability by about 20%. Although changes in platelet factor 4 (PF4) and [3TG are about 30% to 40%, and changes in response to aggregating agents is about 75%, however in the preparation of the sample itself can affect these test results. 4~ In contrast, although changes MPV during the 5 days shelf life are about 20%, the dMPV changes in some types of PC are 100% to 200% by our methodology. 43 Thus dMPV seems to provide a highly sensitive, reproducible, quantitative measurement of the PSL and can be carried out, in a standardized manner, on a sample obtained nondestructively from the connecting line of the bag. There is a good correlation between the various tests of platelet functions and dMPV (Table 5). This new and simple procedure has enabled us to eliminate the need for the tedious and cumbersome conventional functional tests for the PSL and/or tests based on the expression of activationdependent markers119,43

138

SEGHATCHIAN AND KRAILADSIRI

Table 5. Relationship Between Various Laboratory Parameters of Platelet Concentrates During Storage r 20 IJM ADP v dMPV* 10 pM ADP + 100 pg/mL collagen v dMPV pH v dMPV Days 0-7 Days 3-7 HSR v dMPV vWF:Ag v dMPV 13TG v dMPV LDH v dMPV dMPV v age 20 pM ADP (%) v age 10 pM ADP + 100 pg/mL collagen v age pH v age Days 0-7 Days 3-7 HSR v age vWF:Ag v age 13TG v age LDH v age GC v age GC v dMPV GC v [3TG GC v vWF vWF v 13TG

p

0.81

0.0436

0.81

0.0414

0.76 1.00 0.85 -0.92 -0.96 0.95 -0.98 -0.78

0.0670 0.0002 0.0250 0.0070 0.0013 0.0029 0.0002 0.0600

-0.80

0.0488

-0.76 -0.94 -0.91 0.97 0.98 0.95 0,84 -0.73 0.74 0.54 0.61

0.0670 0.0760 0.0080 0.0080 0.0004 0.0027 0.0010 0.0100 0.0010 0.0100 0.0010

*dMPV was'carried out with a Technicon H * I cell counter.

Released and Releasable vWF

Abnormalities in platelet yon Willebrand Factor (vWF) have been associated with hemostatic dysfunction. 48-49 Some of the characteristic properties of vWF make this protein an ideal marker for establishing the nature of the events involved in development of the PSL, (ie, activation, release reaction, lysis and proteolytic fragmentation). These are summarized below: 1. vWF is an adhesive molecule which is involved in both adhesion and aggregation by linking platelets to platelets, and platelets to endothelium. 49 2. The active release of vWF can occur subsequent to stimulation with trace amount of ADP and thrombin, produced during collection, processing and storage. Moreover, the rate of vWF:Ag release is related to both the type of PC preparation as well as the activation state of the platelets 48,49 3. High centrifugation gravity force (40000g) of supernatant plasma of stored PC leads to a decrease in vWF:Ag level, suggesting the

presence of both soluble and microvesiclebound vWF:Ag. 49 . Released vWF binds to the platelet surface and then is cleaved by calpain and/or other proteases. 48-52A simple ELISA assay for both vWF:Ag and its collagen binding activity (vWF:CBA) is available, helping in the assessment of activity states of vWF:Ag. 49 . Storage-induced reduction of GPIb and GPIIb/ IIIa impairs the binding of vWF to the platelet membrane, therefore influencing platelet function.34,48 . The increased level of vWF:Ag in plasma correlates inversely with the level of vWF:Ag in the remaining platelets, measured in platelet lysate. Moreover, the released vWF:Ag has reduced collagen binding activity, suggesting partial proteolytic fragmentation. 49This is supported by finding that the ratio of vWF:CBA versus vWF:Ag in supernatant plasma decreases during storage. 49 . Quantitative flow cytometric procedures, which differentiate intact platelets from microvesicle bound vWF:Ag, is now becoming available. In some laboratories the use of monoclonal antibodies directed to ristocetin cofactor binding site versus polyclonal vWF enables the accurate definition of possible involvement of vWF proteolysis in the evolution of the PSL. 49 Shear stress is also capable of modulating the vWF molecule by enhancing its susceptibility to proteolytic cleavage leading to a decrease in multimeric forms. 52 . Finally the increased level of releasedvWF:Ag during storage correlates significantly with the result of other functional tests for PSL (Table 5). Thus the assessment of the activity state of vWF, which reflects the adhesive properties of stored platelets provides a unique marker of PSL as well as enabling the differentiation of platelet lysis from proteolytic cleavage.49-52 ,

The Presence o f Soluble Platelet Glyeop~otems in P l a s m a

Quantitative and qualitative abnormalities in platelet glycoprotein expression, eg, GPIb, GPIIb/ IIIa, have been associated with platelet dysfunction. 53,54The activation of platelets during preparation and storage also leads to enhanced expression

THE PLATELET STORAGE LESION

of several glycoproteins on the platelet membrane which are then either internalized or cleaved by various proteolytic enzymes including calpain, thrombin, plasmin, and elastase. 49,52Thus in assessing the PSL, the measurement of the level of platelet-derived glycoproteins in supernatant plasma of platelet concentrate is as important as the measurement of their expression on the platelet membrane by flow cytometry, with and without stimulation by selective agonists ie, ADP, thrombin, Ca-ionophore, and some Ca-chelating agents such as EDTA. We have recently reported that total glycocalicin (GC), a major component of GPIb level in supernatant of PC is relatively low (below 3-4 ~tg/ml on day 1) and may reach, under normal storage conditions, approximately 10 to 15 ~tg/ml at the end of shelf life. 55,56Residual leucocytes in PRP-PC (present at concentration of 0.35 to 0.39 • 109/per unit) accelerate the rate of GC generation by 10-fold. 57 This encompasses both soluble and microvesicle-bound GC in equal amount. Not all platelet derived glycoproteins seem to be distributed in the same proportion of soluble and microvesicle-bound forms. Under normal platelet storage conditions, approximately 50% of GC but less than 20% of GPIIb/IIIa appears in soluble form. 57 The release of GC correlates well with various tests of platelet function (Table 5). Thus the expression of the platelet activation markers GPIb, GPIIb/III, GMP140 and other glycoproteins on the platelet surface, in conjunction with the quantitative measurement of soluble and microvesicle bound forms in the supernatant of platelet concentrates during storage, remains the focus of current interest for the assessment of the nature of PSL.

Quantitation for the Presence of Membranous MicrovesicIes A variety of events lead to microvesiculation of platelets during storage (Table 6). Although it has been difficult to quantitate microvesiculation in PC in a reproducible manner it is clear that microvesicles of different sizes and functions appear in abundance in PC prepared and stored using standard protocols. 58-61 Moreover, microvesicles may be present 'in a higher concentration in certain pathophysiological states and subsequent to development of some BRM, in particular activated complements and platelet antibodies. 62 Of particular relevance to hemostasis is the

139 Table 6. Events Associated with Platelet Microvesicle Formation 9 Mechanical disruption and excessive shear force, ie, passage through low gauge needles or leucocyte filters, high g force, rigorous agitation and metabolic exhaustion. 9 Activation by platelet aggregation agonists, ie, ADP, thrombin, generated during collection and storage. 9 Activation/secretion caused by poor handling and prolonged exposure to cold (4~ and pH (>7.6). 9 Platelet lysis caused by freezing-thawing, lyophilization/ rehydration, and prolonged exposure to low pH (<6.2). 9 Exposure to activated complement components, leucocyte and platelet derived enzymes and metabolites such as calpain, cytokines, histamine.

development of platelet procoagulant activity in the supernatant of PC during storage which is indicative of the release of non-sedimentable membranous microvesicles, with platelet factor 3 (PF3) activity. The level of PF3 is significantly higher in standard PRP-PC comp~ed with apheresis PC. 65 The fact that the level of PF3 decreases to half but not completely abolished by the addition of platelet activation inhibitors such as PGE1, theophylline, and aprotinin, suggests that mechanisms involved in microvesicle formation are multifactorial. 65 Several methods for the quantitation and characterisation of platelet microvesicles are now becoming available and summarized in Table 7. In practice, flow cytometry is the current method Table 7. Available Methods for Qualitative/Quantitative Evaluation of Platelet Microparticles 9 Change in platelet size distribution pattern and cellular indices by automated cell counters. 9 Immunoelectrophoresis using 1251labelled monoclonal antibodies specific to platelet membrane glycoproteins. 9 Direct labelling of microvesicles with 1111nand/or binding of the labelled annexin V to assess the degree of PS exposure. 9 Flow cytometric analysis activation and/or secretion specific markers fie, GPIb, IIb/lla, GMP-140, thrombospondin, vWF). 9 ELISAto differentiate soluble and microvesicle bound markers from smaller cellular fragments based on differential centrifugation or passing through filters with small pores. 9 Indirect effect of microvesicles on other tests reflecting platelet adhesion, aggregation and correction of the bleeding time in vitro fie, clot signature analyser). 9 Regulation of thrombus formation through prothrombinase and protein C pathways in the presence of other subcomponents of bioamplification/bioattenuation processes. 9 Indirect effect on adhesion of platelets to subendothelium in the Remuzzi and Baumga~tner apparatus.

140

SEGHATCHIAN AND KRAILADSIRI

of choice as it allows direct enumeration of microvesicles and the activation state of remaining platelets. At the same time microvesicles can be differentiated from platelets by their side scatter and fluorescence intensity. 65,66 Flow cytometry can be carried out even without a plasma separation step, therefore minimise, the centrifugation-induced platelet activation, release reaction and microvesiculation. Various monoclonal antibodies (MoAbs) are now available for the estimation of microvesicles derived from platelets and other cellular contaminants helping in this identification as well as in identifying the mechanism of microvesicle formation. Of particular relevance are the markers that are related to platelet phosphatidyl serine (PS) exposure; eg, MoAbs specific to various coagulant or anticoagulant factors bound to the platelet membrane such as FVa, FVIIIa, FIXa, FXa, IIa, protein C and protein S. Apart from prothrombinase, the use of fluorescein-conjugated annexin V, a new marker, tenase, protein C/sases subcomponents, that can be applied for detection of microvesicles. Annexin V is known to compete with factor V binding sites on platelet phosphatidyl serine with high affinity and high specificity, hence producing an anticoagulant effect. 66

TRANSFUSION REACTIONS AND THE PLATELET STORAGE LESION

The transfusion of PC brings about an almost instantaneous therapeutic benefit, but it also can initiate ~t number of changes in the recipient. Most of the available PC preparations show some evidence of activation but to what degrees these activated platelets continue to circulate by avoiding the in vivo clearance processes is poorly understood. It is generally accepted that about a third of transfused platelets are retained in the spleen for several hours, and are released into the circulation within 6 hours. 62 During this period it is assumed that the sequestered !platelets undergo the process of rejuvenation and regain their functional capacity as indicated by the shortening of the bleeding time. In a recent study on three types of PC, we have observed that platelets derived by three different methods (PRP-PC, BC-PC, Cobe Spectra PC) are heterogeneous with respect to their in vivo response and production of transfusion reactions. Although all three types of PC showed approximately the same increments at 24 hours, Cobe

Spectra PC were found to be superior on the basis of 1 hour recovery and had the lowest frequency of transfusion reactions (Table 8). These differences may be related to different facets of the PSL including the platelet lesion induced by the different types of container; bag surfaces and/or tubing, possibly through metabolic exhaustion and generation of lactic acid, generation of cytokines and activated complement components 67 leading to generation of microvesicles. All of these factors may be responsible for the observed differences in the three types of PC in terms of both reduced transfusion efficacy and increased rate of transfusion reactions. (Table 8) There is now ample evidence that platelet derived microvesicles share many of the biological capabilities of intact platelets. However to what degree microvesicles contribute either directly or indirectly to the hemostatic effectiveness of the transfused platelets or to what degree they might attribute to some of their harmful effects remains to be fully established. Even if microvesicles produce a desired hemostatic effect in some groups of patients, the half-life of microvesicles in vivo is likely to be short. Moreover, because of high expression of PS on their surfaces and because of high affinity of PS for several plasma proteins the formation of microvesicles might be responsible for the expression of neoantigens which could modulate some immunological reactions in recipients. On the other hand, PS expression on microvesicles may contribute to the bioattenuation process through protein C anticoagulant activity in the Table 8. Comparative Analysis of Corrected Count Increment (CCI) and Transfusion Reactions with 3 Types of Platelet Concentrates* Products

lhCCI 1 hCC1->7.5 24hCCI 24h'CC1>-4.0 TxReactions Mean % Mean % %

PRP-PC (n =' 117) 7.16 BC-PC (n = 169) 7.27 Cobet (n = 169) 9.08

56

4.81

50

17.1

67

6.11

54

4.1

76

7.26

54

3.6

*Indication for transfusions: 96% prophylaxis and 5% to correct bleeding. The overall transfusion reaction rate was significantly lower (P < 0.005) in both BC-PC and Cobe Spectra PC cbmpared with PRP-PC. tCobe Spectra PC showed significantly improved recoveries at 1 h ( P < 0.005) but not at 24 h. No acute reactions were associated with this product. Modified arid reprinted with permission. 21

THE PLATELET STORAGE LESION

microvasculature where thrombomodulin is pres~ ent in abundance. 68 Thus it remains unclear to what extent the presence of microvesicles in PC might modify the hemostatic balance between hypercoagulability leading to thrombosis, or bleeding. Platelet concentrates also contain a substantial amount of other cellular contaminants such as viable granulocytes, lymphocytes and red cells (0.1 to 2.5 ml/unit) as well as fragmented and dead platelets. Of particular significance to the PSL and the production of transfusion reactions is the role of leukocytes as leukocytes both accelerate the formation of the PSL and contribute to adverse reactions in recipients. Moreover, their removal, in particular with the use of a negatively charged filter can cause cellular injury, affecting platelet storage stability as well as activating the kallikrein/kinin system causing anaphylactic shock in some recipients. 69-72 Some of the above mentioned BRM are preferentially reduced with the use of positively charged leucocyte filter. 73 Thus, knowledge of the differential diagnosis of transfusion-associated complications remains a challenging area of transfusion medicine. The protein burden in plasma is even more diverse. In addition to modified constituents, there are substances secreted, or peeled off from the cells such as soluble HLA antigens. There is the poSsibility of extending the shelf life of stored PC, through cryopreservation, but with deleterious effects. As cryopreserved PC must be washed repeatedly to remove the cryoprotectant agent before transfusion. Lyophilized para-formaldehyde treated-PC can be produced effectively with a similar morphology to washed or 5 days stored PC, having the same expression of GPIb and G P I I b / I I a on their surface. v4 Nevertheless in vivo studies are needed to assess their in vivo effectiveness as well as the transfusion reactions associated with such products. The effectiveness and the therapeutic role of other soluble, microvesicle or surface-bound hemostatic components also remain to be elucidated. Thus the optimisation of storage stability of PC still constitute a challenging area in transfusion medicine. CURRENT AND FUTURE TRENDS

Platelet therapy is an area of modern transfusion where Objective criteria rather than subjective impression is the preferred mode of clinical decisionmaking. However in line with the concept that "the

141

pureris better" newer strategies have been used for reducing refractoriness and febrile reactions ie, by the better selection of donors and improved preparation and storage of platelets to minimize platelet injury. The relevance of the in vitro changes to in vivo function stillremains unresolved in m o s t instances. Ideally the least changes that occur during storage, the closer, a given platelet preparation can be considered to reflect the native subpopulation of functional platelets. Meanwhilel the search for the mechanism of platelet activation/storage lesion will continue as will methods to produce PC which are viral and bacterial inactivated, v5 The application of ex vivo expansion, and the use of recombinant monoclonal purified products for selective depletion or enrichment of platelet products, may also reduce the demand for purified PC. In the near future, lyophilized or stabilized dried platelets may become a practical reality. TM Such PC would have far less demanding storage conditions and could be reconstituted in situ with suitable buffers before transfusion. Moreover with the likely availability and use of cloned recombinant thrombopoietin (r-TPO) to improve the rate of platelet recovery in thrombocytopenic conditions the recent increasing requirements for PC may start to decline. A great deal has been learned about the numerous changes that occur during the storage of platelet and their correlation with in vivo function. The increasing sophistication in processing of blood components to improve quality standards, coupled with the implementation of newer technologies and products, are essential for continual improvement. It is likely that we have only begun to glimpse the role of the PSL in producing the long term adverse effects that may result from the transfusion of stored PC which have undergone the storage lesion. In conclusion, it seems that the PSL occurs through a sequelae of events involving shape change, expression of activation specific markers, loss of functional membrane integrity, and microvesiculation or fragmentation through increased metabolic activity associated with lactate accurriulation. Although excellent methods for the estimation of platelet injury are available, there is some doubt as to what degree each of these assays reflect the performance in vivo of injured platelets. These assays are nonetheless quite useful, in the development of a new protocol and/or product, or in

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SEGHATCHIAN AND KRAILADSIRI

examining the impact of a major variation of old product/process. Finally such tests will enable a better understanding of the PSL. For the purpose of routine monitoring of the PSL, in process validation programmes and the prerelease testing of platelet concentrates, it is our belief that a simple and practical test, based on morphological or functional integrity and state of platelet activation/ aggregation measured by paired sampling (dMPV/ dPLT) is of both diagnostic and prognostic values. This approach will also offer an unbiased objective quantitative statistical approach to assessing platelet viability as well as predicting the functional

integrity of platelets in PC. Importantly, such procedures are also amenable to large scale screening and virtually complete automation, allowing prerelease testing of platelet function.22,43 ACKNOWLEDGMENT The authors are grateful to Drs B Brozovic and A Bode for providing many fruitful comments and to our longstanding collaborators at NIBSC, in particular Dr Wadhawa for the cytokine assay and to Andy Miller for help in the graphics as well as to members of the Quality Department for Data Collection and Office Services in particular Joanne Foley for preparation of this manuscript.

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THE PLATELET STORAGE LESION

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disorders and congenital thombophilia, CRC Press, Boca Raton, Florida 1996, pp 2-17 69. Adamson JW, Hollinger FB, Snyder EL, et al: Challengers to transfusion medicine: Infectious and Immunologic complications of blood transfusion. Hematology 1995, Education Program American Society of Hematology (Seattle, Washing~ ton) 1995, pp 82-92 70. Miletic VD, Popovic O: Complement activation in stored platelet concentrates. Transfusion 33:150-154, 1993 71. Krailadsiri E Seghatcbian MJ: Negatively charged filters significantly enhanced kallikrein and ttuombin-like activities in platelet concentrates. Thromb Res 83:469-474, 1996 72. Sano H, Koga Y, Hamasaki K, et al: Anaphylaxis associated with white-cell reduction filter. Lancet 347:105, 1996 (letter) 73. Shimizu T, Uchigiri C, Mizuvo S, et al: Absorption of anaphylatoxins and platelet specific proteins to filtration with a polyester leucocyte reduction filter. Vox Sang 66:161-169, 1994 74. Read MS, Redduck RL, Bode AE et al: Preservation of hemostatic and structural properties of rehydrated lyophilised platelets: Potential for long-term storage of dried platelets for transfusion. Proc Natl Acad Sci USA 92:397-401, 1995 75. Lin L, Londe H, Janda JM, et al: Photochemical inactiva~ tion of pathogenic bacteria in human platelet concentrates. Blood 83:2698-2706, 1994