In Vitro Assessment of the Quality of Stored Platelet Concentrates

In Vitro Assessment oC the Quality oC Stored Platelet Concentrates Scott Murphy, Paulo Rebulla, Francesco Bertolini, Stein Holme, Gary Moroff, Edward Snyder, and Robert Stromberg for the BEST (Biomedical Excellence for Safer Transfusion) Task Force of the International Society of Blood Transfusion

N A RECENT editorial, Snyder indicated that one of the "Holy Grails" in transfusion medicine is to find a simple in vitro assay that could be performed on platelets prepared for transfusion and would predict their in vivo reeovery, survivaI, and function. l The purpose of this review is to summanze the data published in this area since 1980. The goaI is to identify those assays for which elear correlations with in vivo results have been established. The principles behind these assays are discussed so that correlation can be made between anatomie and biochemical ehanges in pIatelets and loss of in vivo effectiveness. It is hoped that this information can be used to define assays that could be used for quality controi proeedures and to indicate those that might be used by manufacturers and blood eenters during development of new methods of preparing and storing platelets for transfusion. Before proceeding with the formal literature review, it is necessary to review less formally the literature from the late 1960s and 1970s, because some of the assays were developed and validated prior to 1980. During this earIier period, it was shown that platelets stored at 22°C were hemostatically more effective in vivo than those stored at 4°C. 2-4 Furthermore, when platelets were stored at 22°C in containers available at that time (firstgeneration containers), there was frequent1y a decrease in pH to below 6.2, which was associated with irreversible morphological changes and loss of in vivo effectiveness. 5 Throughout this review pll measurements refer to those made at 22°C. Finally, during that period many laboratories were studying methods of storing platelets by freezing. 6.7 Two assays used during this period correlated well with in vivo results as judged by autologous, radiolabeled reinfusion studies in healthy volunteers. The first was the response of platelets to hypotonic stress or shock. 6-8 Another widely used descriptive term for this assay, the osmotic reversal reaetion, will be used in this review. This assay, first described by Fantl,9 is based on the fact that the light transmission through platelet-con-

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Transfllsion Medicine Reviews, Vol VIII, No 1 (January). 1994: pp 29·36

taining plasma increases abruptly after the addition of hypotonic solutions, because the influx of water into the eell leads to swelling and a dilution of cytoplasmic contents. The refractive index of intraeellular fluid decreases, leading to increased light transmission. However, "healthy" cells "prefer" to regain their initial volume and initial concentration of cytoplasmic colloids. Water and electrolytes are gradually pumped out, and there is a paralIel return of light transmission toward baseline. It was shown that there is correlation between failure of this reversal and loss of in vivo viability caused by storage at 4°C, exposure to pH less than 6.2, and freezing injury.6-8 The second assay described during this period was assessment of platelet morphology by oilphase microscopy. Kunicki et al 10 described a morphology score method that has been widely used since its description in 1973,u The percentage of eaeh morphological type is multiplied by a series of arbitrary factors: disks X 4, spheres X 2, dendritic forms XI, and ballooned forms x O. It was generally recognized that storage at 4°C, exposure to pll less than 6.2, stimulation with aggregating agents, and freezing injury aU converted plateIets from their characteristic diskoid shape to a spherical form. It was shown that recovery and survival in vivo correlated with the morphology score when storage at 4°C l l and in first-generation From the Cardeza Foundation for Hematologie Researeh. Jefferson Medieal College, Thomas Jefferson University Hos· pital. Philadelphia. PA; Ospedale Maggiore Di Milano. Insti· tuto Di Rieovero E Cura A Carattere Scientifieo, Centro Tras· fusionale E Di Immunologia Dei Trapianti, Milan. Italy; Ameriean Red Cross, Mid·Atlantie Region and Eastern Vir· ginia Medieal Sehool. Norfolk. VA; Jerome H. Holland Labo· ratory. Ameriean Red Cross, Roekville. MD; and the Depart. ment oj Laboratory Medicine. Yale University Sehool oj Medicine, Yale New Haven Hospital, New Haven, CT. Address reprint requests to Scott Murphy, MD, Cardeza Foundationfor Hematologie Researeh. Jefferson Medieal Col· lege. Thomas Jefferson University Hospital, /015 Walnut St, Philadelphia, PA 19107. Copyright © 1994 by W.B. Saunders Company 0887·796319410801·0003$3.0010

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containers 10 were evaIuated. In retrospect, this is not surprising, because as mentioned above, storage under these conditions causes cellular injury and associated disk-to-sphere transformation. There has never been verification that inclusion of dendritic and ballooned forms in the classification adds to a simpler assessment of relative percentage disks and spheres. It has been the authors' experience that dendritic forms are common earIy during satisfactory storage at 22°C. However, there is no direct evidence available that indicates that such forms are less effective in vivo than spheres, Iet aIone disks. The point to stress is that these assays were deveIoped and validated during the study of methods of storage that are no Ionger being used. PIateIet concentrates are now stored at 22°C in secondgeneration containers l2-14 that allow adequate oxygen and carbon dioxide exchange l5 so that a pH decrease shouId not occur. It shouId be stressed that the capacity of second-generation containers for gas transport can be exceeded if an excessive number of platelets are stored in them. 16 Furthermore, even with adequate gas exchange, pH may decrease during storage of platelets in an additive solution if the solution has inadequate buffering capacity. 17.18 Thus, current in vitro measurements must be sensitive to the lesions produced by current methods of preparation and storage as well as those produced in first-generation containers.

Table 1. Assays Used to Assess Platelet Quality

Assay

pH Platelet eount Aggregation in response to agonists Osmotie reversal reaction

PO, Pco z Morphology by phase mieroscopy Supernatant lactate dehydrogenase release Supernatant beta-thromboglobulin and platełet faetor 4 rełease Supernatant lactate concentration Supernatant glucose concentration Leukocyte concentration Mean platelet volume Platelet serotonin content or release into supernatant Platelet adenosine triphosphate content Morphology by electron microscopy Platelet size distribution Platelet glycoprotein Ib expression Extent of shape change in response to agonists Platelet adenine nucleotide content Disk shape assessed objectively* Platelet glycoprotein IIb-llla expression Supernatant thromboxane-B 2 content PlateJet P-selectin expression Swirling of coneentrates assessed by visual inspeetion

Number ot Reports in Which Cited

127 118 81

57 54

52 51

50 48

44

39 36 29

26 20 19 17

16 15 12

10 10 9

8 4

* That is, not by microscopy.

MATERIALS AND METHOOS

We used the Medline (US National Library of Medicine) system produced on optical disks by Silver Platter Information, Inc (Norwood, MA). All articles published from January 1980 to September 1992 containing the words platelet concentrate or platelet transfusion in the abstract were examined. Criteria for further evaluation were: (1) publication in the English language, (2) presence of a materials and methods section, (3) studies involving human platelets, (4) conditions of platelet preparation and storage applicable to clinical transfusion, and (5) platelet storage in the liquid state at ambient temperatures using containers designed to provide enough oxygen entry to insure against decrease in pH. RESULTS

Atotal of 199 articles were found that conformed to the above criteria. Table 1 lists the 25

most commonly used assays in order of frequency of use. Table l indicates that a wide variety of measurements have been used, presumably as reflections of platelet quality and, therefore, as predictors of in vivo efficacy. The 199 articles were then reexamined to find those that described in vivo assessment of platelet quality. Fifty-two articles were found. Because we had excluded those studies in which a significant number of concentrates exhibited a decrease in pH because of the use of a first-generation container, the earliest study included was published in 1982,12 so that we essentially reviewed 10 years of experience. Of the 52 articles, 13 had too little in vitro data to allow meaningful comparisons with the in vivo data provided. Of the remaining 39 articles 17 included in vivo measurements that indicated that the platelets being studied had good in vivo efficacy. 16,17,19-33 These articles allowed a

IN VITRO ASSESSMENT OF PLATELET CONCENTRATES

description of the in vitro characteristics of platelets that had been prepared and stored with good in vivo characteristics, but did not provide information conceming the characteristics of plateIets that performed badly in vivo. There were 22 articles that provloded both . 12-14,18,34-51 Examination of these 39 reports allowed us to describe the characteristics of platelet preparations that had adequate in vivo performance. The majority of these in vivo studies were autologous, radioisotopic reinfusion studies in healthy volunteers. Some involved measurement of increments in platelet count after transfusion of thrombocytopenic recipients. Adequate in vivo performance is defined in this review as recovery and survival that are equivalent to or better than platelets stored for 5 days at 22°C in second-generation containers. In some assays, plateIet characteristics change very little during adequate preparation and storage. For example, mean platelet volume either stays the same or decreases by 10% or less. ReIease of lactate dehydrogenase is approximately 10% and rarely more than 20%. During platelet storage, the ability of platelets to take up serotonin is unaltered and platelet glycoproteins Ub-IIIa and Ib are either stabIe or decrease by less than 15%. It could be predicted therefore that these measurements would be relatively insensitive to platelet damage because platelets stored for 5 days at 22°C in secondgeneration containers elearly have an in vivo recovery and survivalless than that of fresh platelets (perhaps, a deeline to two thirds relative to fresh platelets). On the other hand, platelets stored for 5 days or even shorter intervals at 22°C aggregate very poorly and release very low concentrations of serotonin and adenine nueleotides in response to single platelet agonists such as adenosine diphosphate, epinephrine, and collagen. Therefore, such assays are too sensitive, because platelets with major defects have adequate in vivo performance. It has been shown that well-stored platelets maintain some, although not normal, response to stronger stimuli such as thrombin, ristocetin, and pairs of the weaker agonists such as adenosine diphosphate and epinephrine or adenosine diphosphate and collagen. 52 Other assays show a elear deeline during adequate platelet storage without showing a marked deficit. Typically, the morphology score, the osmotic reversal reaction, and platelet adenosine

31

triphosphate Ievels decrease to approximately 60% of the values for fresh platelets. The reIease of beta-thromboglobulin and platelet factor 4 reaches 30% to 40% during such adequate storage. Therefore, these assays are good putative candidates to have an appropriate degree of sensitivity to assess platelet changes during preparation and storage. The 22 reports in which both good and inferior in vivo results were presented and correlations made with in vitro measurements allow the assessment of in vitro measurements as candidates for predictors of in vivo viability. In three reports I4 ,39,40 Snyder et al studied four storage containers, several forms of agitation of plateletsduring storage, and platelets harvested from granulocyte preparations and compared in vivo results with several in vitro parameters. In all three studies, there was poor correlation between in vivo results and the release of beta-thromboglobulin and lactic dehydrogenase. In fact, in some circumstances, the in vivo results were superior when measurements of lactic dehydrogenase 14 and beta-thromboglobulin40 suggested that they would not be. In two other studies,36,37 significant differenees between experimental and control groups were found for release of beta-thromboglobulin and platelet factor 4, but differences were not found when in vivo studies were performed. Thus, on the whole, the literature does not support the use of assays measuring release of lactic dehydrogenase, platelet faetor 4, or beta-thromboglobulin as predietors of in vivo funetion. In two of the studies of Snyder et al in vivo results eorrelated with morphology scores and the osmotic reversal reaction. 14,40 In two studies during the development of seeond-generation containers, Murphy et al deseribed superior in vivo results from a tumbling mode of agitation versus an elliptieal mode l2 and for storage in a CLX container as opposed to a PL-146 container. 43 In both studies, in vivo superiority correlated with the extent of shape ehange with exposure to adenosine diphosphate and the dispersion of the size distribution as determined by Coulter counter (Coulter Electronics, Hialeah, FL). These two assays had originany been deseribed by Holme et ae 3 in 1978. The extent of shape change is a quantitative assessment of the fact that the addition of an agonist such as adenosine diphosphate or thrombin to a suspension of diskoid platelets stirred in an aggregometer eauses disk-to-sphere transformation, which in tum cor-

MURPHY ET AL

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relates with a decrease in light transmission. In fact, the extent of shape change really assesses the number of diskoid platelets that were present before the addition of the agonist. A suspension of spherical platelets can display no shape change. Therefore, it is really a quantitative approach to assessing the percentage of disks in a platelet population. Its success as a predietor adds further support to the use of the morphology score or at least a mieroscopie assessment of disk percentage. When studied by Coulter counter, platelets display a log normal size distribution. Therefore, not only mean platelet volume but also the dispersion or geometric standard deviation of the size distribution can be measured. During platelet storage, dispersion tends to increase because of the formation of populations of abnormally small and large swollen platelets. 41 As shown by Murphy et al, 12,43 this process can become extreme under adverse circumstances correlating with poor in vivo results, In fact, Holme et al had shown good correlation between extent of shape change, dispersion, and the osmotic reversal reaction and in vivo viability in their original description of these techniques in 1978,53 More recent work from Murphy et al 18 also showed a correlation of the osmotic reversal reaction and in vivo results during studies of a synthetie medium for storing platelets. As mentioned in the previous section, it is well known that platelet viability is lost when pH decreases to 6.2 or belowand that the finding of pH less than 6.8 for platelets stored in plasma suggests exposure to hypoxic conditions. Therefore pH is an important and simple in vitro measurement to use in assessing platelet quality. There is very little information as to whether pH can be tOG high. In one study by Murphy et al 12 of various forms of agitation for platelet storage, poor in vivo viability correlated with an increase in pH to or above 7.6. There is inadequate information in this area, but for the present, this level seems a reasonable one to cause concem. The work of Holme et al, George et al, Heaton et al, and Keegan et a144-51 ,54 has provided a great deal of data which is relevant to these questions. In their initial studies of platelet additive solutions in 1987,44 they found superior in vivo results for the additive solution relative to storage in plasma and a strong correlation between superior in vivo results and higher values for the osmotic reversal reaction and the extent of shape change. Such cor-

relation was confirmed in their subsequent studies for the osmotie reversal reaction48 ,51 and the extent of shape change. 46 ,48,49 Also, in 1987 Holme et al reported a strong correlation during platelet storage for platelet content of adenosine triphosphate with the osmotic reversal reaction and extent of shape change. 54 Therefore, it was not surprising that, in subsequent reports, they found good correlations between superior in vivo results and superior maintenance of platelet adenosine triphosphate levels. 44 ,46-48,51 These investigators have also reported correlations between superior in vivo results and reduction in the rate of lactate production per platelet during storage,44,47-51 and maintenance of oxygen consumption at the completion of storage. 49 ,51 Although these correlations have not been evaluated by other investigators, they suggest that platelet integrity correlates with vigorous oxidative metabolism with minimai reliance on glycolysis. DISCUSSION

This literature review allows for some generalizations conceming the relationship between in vitro tests of platelet preparations and their in vivo effectiveness, In most cases, the in vivo assay reported in these artieles has been an autologous, radioisotopic reinfusion study in a healthy volunteer or, more rarely, the measurement of increments in platelet count in thrombocytopenic patients. Thus, the in vitro assays are being correlated with capacity to circulate after infusion. These in vitro assays cannot be said to correlate with in vivo function. Bleeding times have been measured in thrombocytopenic patients after infusion to assess platelet function. 4,12,13,26 However, the logisties of these studies are difficult and there is concem about the risk of infection in these patients who are usually leukopenie. There is no documentation in the literature that this concem is justified. Nonetheless, the development of methods for predicting in vivo function awaits creative work. With this caveat, we can say that the osmotie reversal reaction and the morphology score (or another measurement reflecting diskoid shape) have stood the test of time since they were found to correlate with in vivo results during the study of platelet storage at 4°C, in first-generation containers, and by freezing. The value of these tests is

IN VITRO A55E55MENT OF PLATELET CONCENTRATE5

further supported by the fact that many laboratories have used them successfully, undoubtedly with individual variations on the methods first described 15 to 20 years ago. Assessment of diskoid shape can be made in several ways. Oil-phase microscopy is the time-tested approach, but it requires a skiIled observer and is subjective. The measurement of the extent of shape change has not been widely used, but it probably accomplishes the same purpose as oil-phase microscopy but in an objective fashion. It requires a platelet aggregometer as does the osmotic reversal reaction. Platelet diskoid shape is also reflected in the "shimmering" appearance of fresh platelet concentrates during visual inspection. Concentrates of spherical platelets do not give this appearance. This simple assessment has not been studied extensively, but it may become useful for routine quality contro!. However, it is subjective and allows only discrimination between concentrates of diskoid and spherical platelets and perhaps one or two intermediate gradings. Three instruments have been developed to make this measurement in an objective manner. 34,55,56 One study45 did not support the ability of two of these instruments to prediet in vivo viability although, for one ,55 there was correlation between the readings from the instrument and platelet morphology. The results with the third instrument56 have not yet been correlated with in vivo results. Other assays have been less widely validated but show promise. Based on the extensive experience of Holme et al 44 ,46-48,51 correlating platelet adenosine triphosphate content and in vivo viability, this measurement has promise for the future. Because it probably reflects a different component of the integrity of the platelet from diskoid morphology and the osmotic reversal reaction, it is a good candidate to be used in a panel of tests along with assessment of diskoid morphology and the osmotic reversal reaction. Other assays that reflect platelet metabolic activity, such as rate of lactate production per platelet, oxygen consumption rate, and the partial pressures of oxygen and carbon dioxide, deserve further work. Although the work of Holme et al44 ,47-51 has shown correlation between rate of lactate production per platelet and in vivo viability, there are situations in which such correlation clearly would not be expected. For example, deleterious forms of agitation may injure platelets severely early in storage,12 resulting in both loss of

33

viability and a decrease in the rate of lactate production. The measurement by Coulter counter of dispersion or geometric standard deviation of the platelet size distribution has also been found to correlate with in vivo viability. Unfortunately, all of the work was performed with one Coulter counter that was manufactured in the 1960s. It has been the experience of two of the authors (S.M. and S.H.) that this correlation has been much less strong with counters manufactured more recently. Damaged platelet preparations that have increased dispersion with the old counter have more normal dispersion with the newer counters. It may be that the newer machines edit out pulses from badly damaged platelets because the pulses seem not to reflect biological material, but this speculation has never been verified. This issue deserves further study because abnormalities in the distribution of platelet size and density41 clearly correlate with in vivo viability. However, this assay cannot be strongly recommended at the present time because it depends so strongly on the counter available to the investigator. Some widely used assays have shown less correlation with in vivo results according to this review of the literature. In fact, in some studies, supematant concentrations of lactic dehydrogenase, beta-thromboglobulin, and platelet factor 4 have actually given misleading results. Therefore, there is less rationale for encouraging their use. The same conclusion can be reached conceming measurement of platelet aggregation and release of serotonin and adenosine triphosphate in response to widely used single agonists such as adenosine diphosphate, epinephrine, and collagen. These in vitro functions deteriorate very quickly in platelet concentrates that can be shown to be effective in vivo. There are other assays that have been used for oniy a short period of time so that there is too little information about them to make a final judgment about them at this time. These include the measurement by flow cytometry of glycoproteins Ib and Ub-lIla and P-selectin. Although there is promise in this area, these assays have less appeal for wide-spread use because of the cost of purchasing a flow cytometer. Furthermore, our analysis suggests that the measurement of glycoproteins Ib and Ub-lIla is relatively insensitive to platelet injury. The same seems to be true for the ability of

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MURPHY ET AL

the platelet to take Up serotonin and for the produetion of thromboxane B2 . Electron microscopy has been vital in our understanding of platelet storage lesions, but it seems to be too subjective and not sufficient1y quantitative for the purposes of quality controI and developmental work. The purpose of this artiele is to review what is in the literature at this point in time conceming the relationship between in vitro testing of platelet concentrates and their subsequent performance after infusion in vivo. This is an evolving field and progress is expected. There is no intention to make reeommendations that will be fixed indefinitely. We have not attempted to discuss the cause or causes of platelet injury during preparation and storage. There is some evidence that injury is eaused by platelet activation. 57 Better understanding of the platelet storage lesion should permit the development of new assays or new variations on old assays that reflect that lesion. Having said this, what seems appropriate at this time? Current quality control procedures for routinely prepared platelet coneentrates inelude measurements of platelet concentrate volume, platelet count, and pH at the completion of storage. Recommended lower limits for these measurements for random donor platelet concentrates are 35 mL, 5.5 x 1010 platelets per concentrate, and pH 6.2. An upper limit for pH would be 7.6. In addition to measuring platelet number, it would also be desirable to make assessment of platelet quality. Because diskoid shape has correlated well with in vivo viability, the assessment of swirling has promise, predominantly because of its simplicity. In a study by the BEST Task Force,58 this assessment was found to be reproducible with good concordance between two technologists in nine laboratories. Furthermore, if the intention has been to make a leukodepleted produet, leukocyte counts should be performed using a technique with a lower limit of accurate detection of approximately 1 leukocyte/mm3 These considerations are summarized in Table 2. Table 2. Methods for Quality Control of PllItelet Concentrates Platelet concentrate volume Platelet count pH at the completion of storage Determination of swirling Leukocyte count if intent is to prepare a leukodepleted product

Table 3. Methods for Development of New Products and Procedures Same as Table 2 Measurements that correlate with viability in vivo Morphology score or percent disks by oil-phase microscopy Osmotic reversal reaction Extent of shape change Platelet adenosine triphosphate level Measurements reflecting platelet activation P·selectin on platelet surface Beta-thromboglobulin Measurement reflecting platelet Iysis Supernatant content of lactic dehydrogenase Measurement reflecting in vitro platelet function Aggregation in response to pairs of agonists Measurements reflecting platelet metabolic activity P02 and PC02 Rate of lactate production per platelet

It is beyond the scope of this review to address in detail the complex statistical question of how many assays should be performed per unit time. The purpose of quality controi is to provide evidence that a produet meets defined standards of quality with a stated level of confidence. The basic assumption is that, although individual measurements differ, the pattem of deviation is stable (constant standard deviation and coefficient of variation) around alimiting average. For example, studies by one of the authors (S.H.) indicate that the total platelet eontent in platelet concentrates in blood centers has a eoefficient of variation of about 30%, about an average of 8 x 1010. Current standards in the United States require that 75% of random donor platelet concentrates have platelet contents greater that 5.5 x 1010 platelets. The number of concentrates that is actually produced and distributed is irrelevant. With this coefficient of variation, it can be shown that a sample size of 60 will give a 95% confidence interval of ±7.5%. Clearly, the required sample size for a given measurement will vary depending on the standard of quality and level of confidence desired and the coefficient of variation for the measurement. Therefore, the number of assessments per unit time required would vary for each measurement. A more extensive panel of tests seems appropriate to add to the above when a new procedure or a significant modification of an old procedure is being introduced during produet development. A minimum test panel might include: morphology seore and/or extent of shape change, osmotic reversal reaetion, and platelet level of adenosine

35

IN VITRO ASSESSMENT OF PLATELET CONCENTRATES

triphosphate. I~ additi~n, meas~rements reflecti~g platelet activatlOn, lySIS, functlOn, and metabohc activity may be included. The determination of p-selectin seems promising as a reflection of activation although enthusiasm for it is blunted by the need for a flow cytometer. Extremely high levels of supematant beta-thromboglobulin may reflect extreme degrees of platelet activation during concentrate preparation and storage. A marked degree of platelet lysis would be reflected in high levels of supematant lactic dehydrogenase. Platelet funcrion, at least in vitro, can be assessed by determining the aggregation response to pairs of aggregating agents such as adenosine diphosphate and epinephrine or adenosine diphosphate and colla-

gen. Metabolic activity can be assessed by determining Po2 , Pco 2 , and lactate level during storage and at the completion of storage. Very high P0 2 and very low Pco 2 would be reflections of failing oxidative metabolism. High rates of lactate production have correlated with poor in vivo viability and mayaIso reflect hypoxic conditions during storage. Very low rates of lactate production may indicate platelet damage during preparation or early in storage. These considerations are summarized in Table 3. Of course, if the procedure or product is totally new or a major variation of old procedures or products, in vivo studies should follow this in vitro panel. Recommended methods have been published. 59

REFERENCES l. Snyder EL: Activation during preparation and storage of platelet concentrates. Transfusion 32:500-502, 1992 2. Murphy S, Gardner FH: Platelet preservation. N Engl 1 Med 280: 1094-1098, 1969 3. Slichter Sl, Harker LA: Preparation and storage of platelet concentrates. Br 1 Haematol 34:403-419, 1976 4. Filip Dl, Aster RH: Relative hemostatic effectiveness of human platelets stored at 4° and 22°C. 1 Lab Clin Med 91:618624,1978 5. Murphy S, Sayar SN, Gardner FH: Storage of platelet concentrates at 22°C. Blood 35:549-557, 1970 6. Kim BK, Baldini MG: The platelet response to bypotonic sbock. Its value as an indicator of platelet viability after storage. Transfusion 14:130-138, 1974 7. Valeri CR, Feingold H, Marcbionni LD: The relation between response to bypotonic stress and the 51Cr recovery in vivo of preserved platelets. Transfusion 14:331-337, 1974 8. Handin RI, Fortier NL, Valeri CR: Platelet response to hypotonic stress after storage at 4°C or 22°C. Transfusion 10: 305·309, 1970 9. Fantl P: Osmotic stability of blood platelets. 1 Pbysiol 198:1-16, 1968 10. Kunicki TJ, TuccelIi M, Becker GA, et al: A study of variabies affecting the quality of platelets stored at "room temperature." Transfusion 15:414-421, 1975 Ił. Becker GA, Tuccelli M, Kunicki Tl, et al: Studies of platelet concentrates stored at 22°C and 4°C. Transfusion 13: 61-68, 1973 12. Murphy S, Kahn RA, Holme S, et al: Improved storage of platelets for transfusion in a new container. Blood 60: 194200, 1982 13. Simon TL, Nelson EJ, Carmen R, et al: Extension of platelet concentrate storage. Transfusion 23:207-212, 1983 14. Snyder EL, Ezekowitz M, Aster R, et al: Extended storage of platelets in a new plastic container. Transfusion 25 :209214, 1985 15. Murphy S, Gardner FH: Platelet storage at 22°C: Role of gas transport across plastic containers in maintenance of viability. Blood 46:209-218, 1975 16. Snyder EL, Stack G, Napychank P, et al: Storage of pooled platelet concentrates. Transfusion 29:390-395, 1989

17. Gulliksson H, SalIander S, Pedajas I, et al: Storage of platelets in additive solutions: A new method for storage under sodium cbloride solution. Transfusion 32:435-440, 1992 18. Murpby S, Kagen L, Holme S, et al: Platelet storage in synthetic media lacking glucose and bicarbonate. Transfusion 31:16-20, 1991 19. Hogge DE, Thompson BW, Schiffer CA: Platelet stor· age for 7 days in second-generation blood bags. Transfusion 26:131-135, 1986 20. Adams GA, Swenson SD, Rock G: Survival and recovery of human platelets stored for five days in a non-plasma medium. Blood 67:672-675, 1986 2ł. Pietersz RNI, Loss JA, Reesink HW: Survival in vivo of platelets stored for 48 hours in the buffycoat at 4°C compared to platelet ricb plasma stored at 22°C. Blut 54:201-206, 1987 22. Read EJ, Kodis C, Carter CS, et al: Viability of platelets foHowing storage in tbe irradiated state. Transfusion 28 :446450, 1988 23. Rock G, Tittley P, McCombie N: 5-Day storage of single-donor platelets obtained using a blood ceH separator. Transfusion 29:288-291, 1989 24. Holme S, Ross D, Heaton WA: In vitro and in vivo evaluation of platelet concentrates after cotton wool fiłtration. Vox Sang 57:112-115, 1989 25. Rock G, Sherring VA, Tittley P: Five-day storage of platelet concentrates. Transfusion 24:147-152, 1984 26. Bertolini F, RebulIa P, Riccardi D, et al: Evaluation of platełet concentrates prepared from buffy coats and stored in a glucose-free crystalloid medium. Transfusion 29:605-609, 1989 27. Rock G, Senack E, Tittley P: 5-Day storage of platelets colIected on a blood ceH separator. Transfusion 29:626-628, 1989 28. Rock G, Wbite J, Labow R: Storage of platelets in balanced salt solutions: A simple platelet storage medium. Transfusion 31:21-25, 1991 29. Simon TL, Sierra ER, Ferdinando B, et al: ColIection of platelets with a new ceH separator and their storage in a citrateplasticized container. Transfusion 31:335-339, 1991 30. Dzik WH, Cusack WF, Sherburne B, et al: The effect of prestorage white celI reduction on the function and viability of stored platelet concentrates. Transfusion 32:334-339, 1992

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31. Moroff G, Morse EE, Kakaiya M, et al: Platelet viability following storage for 5 days. Transfusion 24:382-385, 1984 32. Snyder EL, Aster RH, Heaton A, et al: Five-day storage of platelets in a non-diethylhexyl phthalate-plasticized container. Transfusion 32:736-741, 1992 33. Pietersz RNI, Loos JA, Reesink HW: Platelet concentrates stored in plasma for 72 hours at 22°C prepared from buffycoats of citrate-phosphate-dextrose blood collected in a quadruple-bag saline-adenine glucose-mannitol system. Vox Sang 49:81-85, 1985 34. Bellhouse EL, Inskip MJ, Davis JG, et al: Pretransfusion non-invasive quality assessment of stored platelet concentrates. Br J Haematol 66:503-508, 1987 35. Menitove JE, Kagen LR, Aster RH: Recovery and survival in vivo of platelet concentrates prepared with prostaglandin El' Transfusion 28:56-58, 1988 36. Pamphilon DH, Potter M, Cutts M, et al: Platelet concentrates irradiated with ultraviolet light retain satisfactory in vitro storage characteristics and in vivo survival. Br J Haematol 75:240-244, 1990 37. Gulliksson H, Shanwell A, Wikman A, et al: Storage of platelets in a new plastic container. Vox Sang 61:165-170, 1991 38. Rinder HM, Murphy M, Mitchell JG, et al: Progressive platelet activation with storage: Evidence for shortened survival of activated platelets after transfusion. Transfusion 31 :409-414, 1991 39. Snyder EL, Pope C, Ferri PM, et al: The effect of mode of agitation and type of plastic bag on storage characteristics and in vivo kinetics of platelet concentrates. Transfusion 26: 125-130, 1986 40. Snyder EL, Ezekowitz MD, Malech HL, et al: In vitro characteristics and in vivo viability of platelets contained in granulocyte-platelet apheresis concentrate. Transfusion 27: 1014, 1987 41. Holme S, Murphy S: Platelet storage at 22°C for transfusion: Interrelationship of platelet density and size, medium pH, and viability after in vivo infusion. J Lab Clin Med 101: 161-174, 1983 42. Lindberg JE, Slichter SJ, Murphy S, et al; In vitro function and in vivo viability of stored platelet concentrates. Transfusion 23:294-299, 1983 43. Murphy S, Holme S, Nelson E, et al: Paired comparison of the in vivo and in vitro results of storage of platelet concentrates in two containers. Transfusion 24:31-34, 1984 44. Holme S, Heaton WA, Courtright M: Improved in vivo and in vitro viability of platelet concentrates stored for 7 days in a platelet additive solution. Br J Haematol 66:233-238, 1987

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45. George V, Holme S, Moroff G: Evaluation of two instruments for noninvasive platelet concentrate quality assessment. Transfusion 29:273-275, 1989 46. Holme S, Moroff G, Whitley P, et al: Properties of platelet concentrates prepared after extended whole blood holding time. Transfusion 29:689-692, 1989 47. Holme S, Heaton A, Momoda G: Evaluation of a new, more oxygen-perrneable, polyvinylchloride container. Transfusion 29: 159-164, 1989 48. Holme S, Heaton WAL, Whitley P: Platelet storage lesions in second-generation containers: Correlation with in vivo behavior with storage up to 14 days. Vox Sang 59:12-18, 1990 49. Heaton WAL, Holme S, Keegan T: Development of a combined storage medium for 7-day storage of platelet concentrates and 42-day storage of red celi concentrates. Br J Haematol 75 :400-407, 1990 50. Keegan T, Heaton A, Holme S: Paired comparison of platelet concentrates prepared from platelet-rich plasma and buffy coats using a new technique with lllln and SICr. Transfusion 32:113-120, 1992 51. Holme S, Bode A, Heaton WAL, et al: Improved maintenance of platelet in vivo viability during storage when using a synthetic medium with inhibitors. J Lab Clin Med 119:144150, 1992 52. DiMinno G, Silver MJ, Murphy S: Stored human platelets retain full aggregation potential in response to pairs of aggregating agents. Blood 59:563-568, 1982 53. Holme S, Vaidya K, Murphy S: Platelet storage at 22°C: Effect of type of agitation on morphology, viability, and function in vitro. Blood 52:425-435, 1978 54. Holme S, Heaton WAL, Courtright M: Platelet storage lesion in second-generation containers: Correlation with platelet ATP levels. Vox Sang 53:214-220, 1987 55. Fratantoni JC, Poindexter BJ, Bonner RF: Quantitative assessment of platelet morphology by light seattering: A potential method for the evaluation of platelets for transfusion. J Lab Clin Med 103:620-631, 1984 56. Jaremo P: Computerised method for monitoring stored platelet packs. Med Biol Eng Comput 26:193-198, 1988 57. Bode AP: Platelet activation may explain the storage lesion in platelet concentrates. Blood Cells 16:109-126, 1990 58. Murphy S, Bertolini F: Assessment of swirling and percent discs by oil, phase microscopy as quality control procedures for platelet concentrates. Transfusion 33:105, 1993 (suppl) 59. Snyder EL, Moroff G, Simon T: Symposium on radiolabeling of stored platelet concentrates. Transfusion 26: l, 1986