Complement Activation during Storage of Single-Donor Platelet Concentrates

Single-donor platelets are stored up to 5 days prior to transfusion. Since contact of plasma to plastic surfaces may lead to complement activation, we investigated whether there is any increase in the complement factors C3a, C4a and C5a in routinely stored single-donor platelet concentrates. C3a levels increased about 40-fold during a 7-day storage. C4a levels also increased with storage time but to a lesser extent. By contrast, C5a levels remained stable throughout this period. ADP- and collagen-induced aggregation was impaired after storage of platelets, indicating severe functional injury. In platelet-poor plasma stored under identical conditions a comparable increase in C3a and C4a concentrations was observed. The loss of platelet function during storage might at least in part be due to the excessive anaphylatoxin concentrations observed.     

Growth Factor Release during Preparation and Storage of Platelet Concentrates

The platelet content of platelet-derived growth factor (PDGF), a mitogen stored in the alpha-granules, was studied during preparation and storage of platelet concentrates (PC) and compared to the growth-promoting activity of platelets, β-thromboglobulin (β-TG) and lactate dehydrogenase (LD). We compared PC prepared from platelet-rich plasma (PRP-PC; n= 10) and from buffy coat. Two different pre-preparation storage periods of the buffy coat were used: 4h (BC-PC:4h; n = 10) and 24 h (BC-PC: 24h; n = 5). The platelet content of PDGF and β-TG was measured by a RIA technique and the growth-promoting activity by incorporation of ³H-thymidine in stimulated fibroblasts. The platelet content of PDGF, β-TG and the growth-promoting activity of the platelets decreased in a similar way during preparation and storage of PRP-PC (31 ±2, 35±2 and 33±7%, respectively, at day 5 of storage; mean ± SEM). The release of LD was minor (3.9 ±0.5% at day 5). At day 1 of storage the platelet content of PDGF was significantly better preserved in BC-PC:4h than in BD-PC:24h (88±2 and 81 ±3%, respectively; p = 0.03). Comparing BC-PC:4h and PRP-PC we found a significantly better preservation of PDGF in BC-PC:4h until day 3 of storage (80±2 and 75±1%, respectively at day 3; p = 0.046). In conclusion the preparation of PC according to the PRP method initially induces a higher loss of PDGF, and hence of the growth-promoting activity, than the BC method.     

Occurrence of the Release Reaction during Preparation and Storage of Platelet Concentrates

To determine the degree of damage occurring during preparation and storage of platelet concentrates, the percent release of B-thromboglobulin (BTG) and percent leakage of the cytosolic protein lactic dehydrogenase was determined sequentially from phlebotomy to the end of storage for 72 h at 20-24 degrees C. The effect of storage temperature, pH, and radiation was also evaluated. The results showed that during preparation of platelet concentrate a large degree of release was found after resuspension of the platelet button formed after the high-speed centrifugation. During storage the percent BTG release increased from 18.1 to 40.2% (p less than 0.05). The percent release seen during storage at 4 degrees C (72 h) was 19.2%, while that seen for platelets subjected to temperature cycling at 4-37 degrees C was 24.9%. Both of these values were significantly less (p less than 0.05) than that seen for concentrates stored at room temperature. A negative correlation between pH and BTG release was found (r = -0.64). Irradiation to 10,000 rad did not induce the release reaction or lactic dehydrogenase leakage. We conclude that the degree of in vitro platelet release is dependent on the preparative manipulations, and gentler protocols for preparation and storage of platelets should be investigated.     

Leukocyte Depletion and Storage of Single-Donor Platelet Concentrates

Background and objectives: Because of widespread use of leukocyte reduction in platelet concentrates (PCs) and the need to store such concentrates, we investigated the effects of leukocyte depletion on the quality of stored PCs. Materials and methods: Ten double-sized PCs were divided into 2 equal units which were tested simultaneously. One half was stored for 5 days after filtration through a polyester filter, the other one was stored unfiltered. Results: The volume of the 10 ‘oversizeD' PCs was 483±40 ml (mean ± standard deviation) and they contained 5.9±1.5 × 10¹¹ platelets and 80±23 × 10⁶ leukocytes. Filtration significantly reduced the leukocyte concentration (168±56/μl before, 6±4/μl after filtration) and leukocyte count (39.9±11.3 × 10⁶ vs. 1.3±0.9 × 10⁶; p < 0.0005). Filtration caused a platelet loss of 16%, the platelet count decreasing not significantly from 2.91±0.75 × 10¹¹ to 2.40±0.94 × 10¹¹ (p = 0.26). After 5 days of storage all parameters of platelet function (platelet aggregation to several stimuli, hypotonic shock reaction [HSR] and platelet retraction), mean platelet volume, and pH and pCO₂ showed no advantage for PCs filtered prior to storage compared to PCs stored unfiltered. Moreover, platelet aggregation on day 5 using 4 agonists at 10 concentrations showed worse results in 4 assays in prestorage filtered PCs (collagen [4 μg/ml: p < 0.05, ADP [0.2 mM]: p < 0.05, ADP [0.3 mM]: p < 0.05, thrombin [0.6 E/ml]: p < 0.05). But there is no convincing trend in all aggregation tests, and HSR, presumably the most useful parameter, was not different on day 5. Conclusions: There is no advantage in terms of improved quality for prestorage leukodepletion of PCs. Taking into account the obvious disadvantages of filtration, such as platelet loss and increasing costs per transfusion, we conclude that pre- or post-storage filtration of single-donor PCs should be done only for patients who have a clear indication for the transfusion of leukocyte-poor blood products.     

Morphological Changes Associated with pH Changes during Storage of Platelet Concentrates in First-Generation 3-Day Container

The platelet injury and loss of viability that has been shown to occur with storage of platelet concentrates (PC) under conditions with increasing or falling pH were examined using scanning and transmission electron microscopy. After storage, samples were taken for measurement of pH value, platelet count and size distribution, release of lactate dehydrogenase (LDH) into plasma, and for SEM and TEM. Increased levels of LDH were observed in PC with pH above 7.3 and below 6.1. In PC with pH above 7.3 this was related to an increased number (23%) of platelets that were lysed or had a swollen disintegrated internal structure (balloons) as seen with TEM. SEM and Coulter counter studies also showed that platelet fragmentation and formation of microvesicles were prominent in PC with pH above 7.3. The electron microscopic pictures confirmed previous suggestions that platelet disc-to-sphere transformation and cytoplasmic swelling occur when pH falls below 6.7-6.8 during storage. SEM studies showed that concomitant with this change, folds and bulky projections appeared on the platelet surface. In PC with pH below 6.1 the morphological change was irreversible with the appearance of more than 90% lysed and balloon platelets. In conclusion, these studies suggest that the loss of viability observed with PC with pH above 7.3 or below 6.1 after storage is related to an increased percentage of lysed and balloon platelets.     

Evaluation of Apheresis Platelet Concentrates Collected with a Reduced (30-ml) Collection Chamber with Resuspension and Storage in a Synthetic Medium

Recently, the CS-3000® Plus Blood Cell Separator with the TNX-6 platelet separation chamber insert has been furnished with a small-volume (30-ml) collection chamber. In this study, a platelet synthetic medium containing glucose and bicarbonate (PSM) was used for resuspension and storage of this highly concentrated platelet product. Eighteen donors participated in a paired study design where each participant donated platelets on two occasions, once following collection in a standard chamber with resuspension and storage in plasma and once following collection in the new chamber with resuspension and storage in PSM. Substantially higher total platelet counts were obtained using platelets collected in the small chamber and stored in PSM as compared to control (4.4±0.9times10¹¹ vs. 3.5±0.9times10¹¹ platelets, p<0.01 by paired t test). After 5 days of storage, PSM-stored platelets demonstrated higher ATP levels, less lactate dehydrogenase in the supernatant and increased lactate production with resulting lower pH at day 5 of storage (6.94±0.15 vs. 7.08±0.09, p<0.05). There were no statistically significant differences of the survival by multiple-hit estimation of PSM-stored as compared to plasma-stored platelets as determined by ¹¹¹In labeling and infusion. A slight decrease in the initial percent recovery with the additive-suspended as compared to suspended plasma cells was noted: 50±8 versus 54±9%, respectively (p<0.05). In conclusion, the CS-3000 Plus/TNX-6 apheresis system with a new reduced-volume collection chamber and an additive solution provides a plasma-poor and highly concentrated platelet product with satisfactory in vivo viability and in vitro functional characteristics after 5 days of storage.     

Storage of Platelet Concentrates at 22{degrees}C

A technique has been proposed which allows easy preparation of plateletconcentrates within a closed, sterile system and maximal utilization of otherblood components. Lifespan studies have been performed to demonstrate theviability of platelets prepared in this manner. Such concentrates can be storedfor as long as three days with adequate maintenance of viability for tranfusionpurposes if certain technical requirements are met. Important among theseare attention to concentrate volume and pH; agitation of the concentrate ishelpful for maintenance of viability. Twelve studies in thrombocytopenic recipients suggest that stored concentrates will be safe and effective in transfusionpractice.

Submitted on September 27, 1969 Accepted on January 2, 1970     

Alternative Fuels for Platelet Storage: A Metabolic Study

We have studied the metabolism of platelets in vitro using washed platelets. Oxygen uptake and fuel utilization were measured. It was found that glucose is never oxidized to any significant extent and is always converted to lactate, regardless of oxygen availability. Oxidative metabolism fuels 70-100% of the ATP turnover, and oxygen uptake is the same whether the platelet is consuming glucose, acetate or only an unidentified endogenous fuel. When acetate is the added fuel, no endogenous fuel is oxidized, whereas the addition of glucose results in sparing of only 8% of endogenous fuel. Preliminary storage experiments using plasma-free media show that an acetate-containing buffered salt solution provided excellent storage conditions and that a medium without any exogenous fuel is better than one containing glucose. Thus we conclude that a successful storage medium should contain minimal amounts of glucose, and an oxidizable fuel such as acetate, in order to supplement the endogenous one.     

Storage of Platelet Concentrates from Overnight-Stored Blood and Overnight-Stored Buffy Coat: In vitro Studies

Platelet concentrates (PCs) were prepared from single buffy coats derived from fresh blood and from blood units stored overnight, as well as from buffy coats that were stored overnight. The platelet yield from overnight-stored buffy coats was similar to that of fresh blood or overnight-stored blood. PCs were stored at 20–24°C and on day 5 of storage, platelet aggregation with ADP was tested both at 37 and 25°C. Stored platelets aggregated better at 25°C than 37°C. The maximal aggregation (10 μM ADP) of stored platelets from overnight-stored buffy coats was 46±23% (n = 30), while that of stored platelets prepared either from fresh or overnight-stored blood was 27±21% (n = 29) and 22±15% (n = 29), respectively. Extracellular lactate dehydrogenase and ammonia levels, as well as elastase activity were similar in stored PCs of different origin. Our conclusion is that PCs prepared from overnight-stored buffy coat might also be suitable for storage and clinical use. In vivo studies are needed to confirm our findings.     

Evaluation of Storage Conditions of Platelet Concentrates Prepared from Pooled Buffy Coats

Five days storage of pooled platelet concentrates (PCs) with high yields often results in a pH fall and poor platelet morphology despite the use of specific containers. In this study we evaluated two techniques for prolonged storage of PCs with high platelet counts, by measuring pH and platelet swirl. In routine procedures, 90 PCs, prepared from five buffy coats, were stored in a single 1-litre PL 732 container. After 5 days storage, 51 PCs with platelet counts below 3.1 times 10¹¹/U showed a pH above 6.8 and optimal swirling scores. 29 of 39 PCs (74%) with yields above 3.1 times 10¹¹/U showed pH values below 6.8 and poor swirling scores. These poor results can be prevented by shortening the duration of storage. PCs with high yields can be stored for 5 days either by dividing the concentrate into two containers or by adjusting the platelet count to 3.1 times 10¹¹/U. In the latter procedure, we reduced the volume of the concentrate and found that of 102 PCs, only 9 showed poor values for pH and platelet swirl. The data clearly indicate the importance of measuring the prestorage platelet count to select the optimal procedure for concentrate storage.     

Preparation and Storage of Platelet Concentrates: II. STORAGE VARIABLES INFLUENCING PLATELET VIABILITY AND FUNCTION

Factors affecting the viability and function of stored platelet concentrates have been investigated in a blood component programme. It was found that platelets could be maintained for up to 72 h without bacterial contamination under the following conditions: (1) surgical skin preparation at venipuncture site; (2) blood collection in CPD or ACD anticoagulant in a closed bag system; (3) centrifugation of PRP at 3000 g for 20 min; (4) storage in Fenwal PL-146, Cutter CL-2383, or McGaw plastic bags; (5) resuspension of the platelet pellet in 70 ml residual plasma; (6) storage at 22+/-2 degrees C; and (7) constant gentle mixing throughout storage. Platelet viability as determined by recovery and survival is largely maintained, as is platelet function measured by template bleeding time. Both viability and function of concentrated platelets stored at 4 degrees C are severely compromised.     

5-Day Storage of Platelet Concentrates in CLX Containers: Effect of Type of Agitation

To determine the degree of platelet damage produced by different modes of agitation during storage of concentrates for 5 days in CLX blood bags, we studied pH, platelet counts, release of LDH and beta thromboglobulin, morphology and osmotic recovery. Platelets were maintained at 20-24 degrees C on elliptical, 6-rpm circular, 2-rpm circular and flat bed agitators. At 72-120 h platelet concentrates stored on the flat bed shaker had significantly lower pH values than units stored on the elliptical or on either of the circular rotators (p less than 0.05). The percent LDH discharged was highest for the units stored on the elliptical rotator (p less than 0.05). Remaining tests of platelet function were not significantly different for concentrates stored on any of the four agitators. Flat bed shakers were unable to resuspend the platelet 'button' which formed after the final preparative centrifugation. Based on our in vitro studies, we conclude that due to problems with low pH values, flat bed shakers may not be optimal for storing platelet concentrates in CLX blood bags and that some other form of agitation should be used.     

Kinetics of Bradykinin Levels during and after Leucocyte Filtration of Platelet Concentrates

Objective: To measure beadykinin levels in platelet concentrates during and after leucocyte filtration. Methods: Platelet concentrates were leukocyte depleted using three different leucocyte-depletion filters selected to represent filters with negative, positive or neutral charge, respectively. Bradykinin levels were analysed before, during and up to 90 min post-filtration. Results: Significant levels of bradykinin were generated when negatively charged leucocyte depletion filters were used. However, we found a high variation between platelet concentrates prepared from different donors as well as within the same concentrate when this was divided into three aliquots. Generated bradykinin was rapidly degraded in the collection bag and no bradykinin was detectable 60 min post-filtration. Conclusion: Bradykinin generation is more pronounced when negatively charged leucocyte removal filters are used. Due to a rapid degradation of bradykinin in the storage bag, pre-storage filtration should minimise the risk of bradykinin related adverse reactions during transfusion with some filters.     

Preparation and Storage of Platelet Concentrates: I. FACTORS INFLUENCING THE HARVEST OF VIABLE PLATELETS FROM WHOLE BLOOD

Plasma and erythrocyte vitamin B₁₂ was measured in apparently healthy Indian lactovegetarians and compared with the healthy non-vegetarians from the same population. Comparison of the mean values for routine haematological parameters revealed no significant difference in various dietetic groups. The plasma vitamin B₁₂ levels were distinctly lower in the lactovegetarians than in the non-vegetarians. However, the mean erythrocyte vitamin B₁₂ levels in different dietetic groups showed no significant difference.     

Stability of Glycoproteins Ib/IX and IIb/IIIa during Preparation and Storage of Platelet Concentrates: Detection by Binding Assays with Epitope-Defined Monoclonal Antibodies and Physiological Ligands

Preservation of the glycoprotein (GP) complexes Ib/IX and IIb/IIIa, because of their role as specific platelet receptors for adhesive proteins, is essential for the haemostatic efficacy of transfusions of platelet concentrates (PCs). We have combined binding assays with epitope-defined monoclonal antibodies, and with the physiological ligands von Willebrand factor and fibrinogen (Fo), to investigate the total expression and functional status of these receptors, in PCs stored for up to 9 days. Routine preparation and storage for up to 5 days have no apparent effect on the surface expression and the functional status of GPs Ib/IX and IIb/IIIa. However, after prolonged storage for up to 9 days we found a 50% decrease in the level of the total and functional GP Ib/IX content of platelets. This finding parallelled a significant rise in plasma glycocalicin, a proteolytic fragment of GP Ib. In addition, long-term storage promoted an impairment in the exposure of GP IIb/IIIa to Fo, and a 50% decrease in Fo binding capacity, without affecting the complex quantiatitvely. Finally, we also noticed a storage-induced increase in the platelet surface expression of GMP 140, and after 9 days of storage there was a rise in mean platelet volume, and a significant reduction in pH levels.     

Factors Influencing Changes in pH during Storage of Platelet Concentrates at 20–24°C1,2

The magnitude of the pH change during platelet concentrate storage at 20–24°C in polyvinyl chloride containers is not determined solely by platelet count per cubic millimeter of plasma, since a wide variation in pH was observed with similar platelet concentrations. In modified platelet concentrates having lost through centrifugation 3–15% of total platelets and 61–92% of residual leukocytes, the pH was maintained at substantially higher levels than in the paired control platelet concentrates. Leukocyte levels do not appear to determine the magnitude of the pH fall. Continuous oxygen ulilization is needed if the pH is to be maintained near 7.0. However, oxygen tension per se is not the factor which influences the extent of pH change. It has been concluded that a specific platelet subpopulation comprising a small proportion of the total platelets in concentrates and having an enhanced capacity to form lactate may be responsible for a major part of the pH reduction which occurs during storage of many platelet concentrates.     

Platelet Storage Lesion in Second-Generation Containers: Correlation with Platelet ATP Levels

The nature of platelet lesion occurring with storage of platelet concentrates (PC) in second-generation containers was investigated using various storage media and storage periods up to 14 days. In CPD-plasma (control medium), the changes which occurred progressively during storage were loss of discoid shape, microscopic platelet aggregate formation, fragmentation and the appearance of disintegrated, 'balloon' forms. By day 14 less than 10% of the platelets were discoid in shape, the platelet count had decreased by 23%, and there was a 5-fold increase in the amount of lactate dehydrogenase in plasma. Associated with this was a decrease in the platelet oxygen consumption rate, D(O2), loss of cellular ATP and extent of ADP-induced shape change, and a decrease in the hypotonic shock response. These parameters decreased at a similar rate, with a 50% decrease (t1/2) at days 7-9. They correlated highly with each other during storage and also with a fall in pH. At day 14 of storage, mean pH was 6.1 +/- 0.3. To evaluate the effect of pH stabilization during storage, 4 mEq sodium bicarbonate was added to PC in CPD-plasma. Although pH maintenance was much improved, 7.2-6.6 during 14 days of storage, the same in vitro lesions developed, although more slowly. The t1/2 of the same parameters was prolonged for approximately two days. When PC were stored in a plasma-free physiologic salt solution whose salt composition was similar to CPD-plasma, the t1/2 of the parameters increased to 11-15 days of storage, although the platelets eventually developed the same in vitro lesions.(ABSTRACT TRUNCATED AT 250 WORDS)