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Background and Objectives The non-paired two-arm study compared the in vitro storage characteristics of platelets suspended as concentrates in either 100% plasma or a mixture of additive solution (SSP+™, MacoPharma, Mouveaux, France) and autologous plasma in a 70:30 ratio over a 14-day storage period. Materials and Methods The buffy coat-derived pooled platelet concentrates were sampled on days 1, 2, 3, 6, 8, 10 and 14 and tests performed to determine platelet morphology, function, metabolism, activation and apoptosis-like activity. Results Swirling remained strong (score = 3) in SSP+™, whilst scores of 1 and 0 were noted for plasma units by end of storage. In contrast to units in plasma, pH levels remained above seven in SSP+™ units, increasing after day 10. Percent positive expression of CD62P was similar in both groups on day 1 (median of 54% and 56% for plasma (n = 13) and SSP+™ (n = 12), respectively), with SSP+™ units showing a more moderate increase in activation after day 10. A progressive decrease in mitochondrial membrane potential was evident in both groups from day 1, whilst annexin V binding was relatively stable from days 1 to 3, with median values remaining below 6%. Subsequent to this, the percentage of platelets binding annexin V increased to approximately 30% by day 14. Conclusion Platelets suspended in a medium of 70:30 SSP+™ to plasma ratio performed at least as well as platelets in 100% autologous plasma for up to 10 days of storage. Further, results are suggestive of an apoptosis-like process being involved in the platelet storage lesion.

Background In Europe, buffy-coat processing allows for the use of platelet additive solutions (PAS). These solutions, however, have long been questioned for their lack of glucose, a potentially essential nutrient for platelet storage. Using a novel, practical, two-part system for incorporation of glucose into an additive solution (PAS-G), this study compares platelet storage in plasma to storage in PAS-G. Study Design and Methods A paired study design of platelet concentrates (PC) were prepared from leucoreduced pools of eight buffy coats (BCP) split into two equal pools, with suspension in autologous plasma, or PAS-G. On days 2, 5, 7 and 9 of storage, samples were tested using standard in vitro platelet parameters. Data were analysed by paired Student's t-tests. Results During storage, PCs in PAS-G maintain a quality profile that is strikingly similar to PCs stored in plasma in terms of platelet activation (CD62) morphology score, swirl, glucose metabolism and pH. However, PCs in PAS-G perform lower (P

New technological developments make it possible to collect red blood cells (RBCs) by apheresis, which allows for better product consistency and has the potential for improved RBC quality. The purpose of these studies was to evaluate the quality and consistency of units of RBCs collected by apheresis using the MCS+(R) machine (Haemonetics Corp., Braintree, Mass., USA).Two studies were performed. In study 1 (n = 10), using containers and CP2D/AS-3 solutions from Medsep Corp. (Covina, Calif. USA), one-unit apheresis RBCs were compared to manually collected RBCs in a random crossover design. In study 2 (n = 12), 6 subjects had one unit collected, while the remaining 6 subjects had two units of RBCs collected with comparison to previously manually collected RBCs from the same donors. Haemonetics containers and solutions were used in study 2.Low RBC volume variability was found for the apheresis collections with a standard deviation of only 6 ml difference between actual and target volumes. Combining the data from the two studies (n = 21 pairs), at 42 days of storage, the apheresis units showed slightly lower hemolysis (0.44+/-0.26 vs. 0.61+/-0.50%), lower supernatant potassium levels (50+/-3 vs. 53+/-3 mEq/l), and improved tolerance to osmotic shock (47+/-3 vs. 49+/-3%) as compared to manual units (p0.05). There was no statistically significant difference in RBC ATP (3.0+/-0.6 vs. 2.9+/-0.5 micromol/g Hb) or in 24-hour percent recoveries (81+/-6 for apheresis vs. 81+/-4% for apheresis red cells). Apheresis RBC quality was not affected by the manufacturer (Haemonetics vs. Medsep) of solutions and containers.RBC units collected by apheresis demonstrated low variability in volume of RBC mass collected, and showed similar RBC properties as compared to manually collected RBCs after processing and after 42 days of storage.

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

The non-paired two-arm study compared the in vitro storage characteristics of platelets suspended as concentrates in either 100% plasma or a mixture of additive solution (SSP+™, MacoPharma, Mouveaux, France) and autologous plasma in a 70:30 ratio over a 14-day storage period.The buffy coat-derived pooled platelet concentrates were sampled on days 1, 2, 3, 6, 8, 10 and 14 and tests performed to determine platelet morphology, function, metabolism, activation and apoptosis-like activity.Swirling remained strong (score=3) in SSP+™, whilst scores of 1 and 0 were noted for plasma units by end of storage. In contrast to units in plasma, pH levels remained above seven in SSP+™ units, increasing after day 10. Percent positive expression of CD62P was similar in both groups on day 1 (median of 54% and 56% for plasma (n=13) and SSP+™ (n=12), respectively), with SSP+™ units showing a more moderate increase in activation after day 10. A progressive decrease in mitochondrial membrane potential was evident in both groups from day 1, whilst annexin V binding was relatively stable from days 1 to 3, with median values remaining below 6%. Subsequent to this, the percentage of platelets binding annexin V increased to approximately 30% by day 14.Platelets suspended in a medium of 70:30 SSP+™ to plasma ratio performed at least as well as platelets in 100% autologous plasma for up to 10 days of storage. Further, results are suggestive of an apoptosis-like process being involved in the platelet storage lesion.

Previous studies with CPDA-1 and Nutricel preserving solutions indicated that red cell properties were affected by small differences in storage temperature. The influence of a 3 degrees C differential on the preservation of the in vitro properties of ADSOL-preserved (AS-1) red blood cells was investigated in a paired study. AS-1 red blood cells were stored at 2.5 and 5.5 degrees C for 42 days. Extent of hemolysis, glucose consumption and pH levels were comparable at the two storage temperatures. Lactate levels were slightly, but significantly higher at 5.5 degrees C. ATP levels were slightly but significantly higher at 2.5 degrees C, only during the later part of the storage period. 2.3-DPG levels were slightly better retained at 2.5 degrees C after 7 days of storage. Holding units of whole blood for either 1 or 8 h at ambient temperature after phlebotomy prior to processing did not influence the types of temperature-dependent changes. The differences as a function of storage temperature were small and appear to be of no practical importance in connection with the storage of AS-1 red blood cells.

Platelet concentrates stored for 5 days under currently optimal conditions at 22 C demonstrate approximately a 20-30% loss of viability as determined by CCI measurements in thrombocytopenic patients and radiolabeling studies with healthy volunteers. The functionality of platelets is substantially lost during storage. This loss appears, for most part, to be reversible upon infusion. In view of this, stored platelets should be acceptable for prophylactic treatment of thrombocytopenic patients in whom immediate hemostatic function is not critical. However, whether stored platelets are functionally adequate for immediate arrest of perioperative bleeding in surgical patients remain uncertain. Currently, a fully adequate and appropriate test that evaluates the hemostatic function of a stored platelet product is not available. In view of this, a desired goal in research and development of new platelet products may then be to attain a platelet product that is similar to fresh platelets in various haemostatic functions. Whether this is an obtainable or relevant goal remains to be seen.

Although platelets stored by cryopreservation are effective in hemostasis, they acquire a number of functional defects during storage and preparation for transfusion. In addition to known acquired defects such as defective aggregation, decreased resistance to hypotonic shock, and disc-spherocyte transformation, we have shown that cryopreserved platelets have decreased capacity to adhere to subendothelium, compared to liquid-stored platelets. To investigate this decrease in adhesive capacity of cryopreserved platelets, we measured the major adhesive membrane glycoprotein, GPIb, and the principal aggregatory protein, GPIIb/IIIa, using flow cytometry in fresh platelets or in platelets cryopreserved in 5% DMSO. We also analyzed aggregation of cryopreserved platelets or liquid-stored platelets in response to ristocetin as another measurement of GPIb functional capacity. We found that approximately 15% of cryopreserved platelets lost surface-bound GPIb, while there was no measurable loss of GPIIB/IIIa during cryopreservation. The cryopreserved platelets also showed a significant decrease in aggregation to ristocetin, but no loss of response to the stronger agonist, thrombin. The loss of surface GPIb from cryopreserved platelets was modest in degree, approximately that reported for liquid-stored platelets, and does not seem great enough to account for the observed functional changes in aggregation and adhesion.

The addition of platelet activation inhibitors to the anticoagulant and the replacement of plasma with a fortified electrolyte medium have been shown separately in previous work to improve the storage of platelets during a 2-week period. In the present study, we have combined these strategies to investigate whether a synergistic improvement could be obtained. A total of 85 concentrates was studied with 300nM prostaglandin E1 (PGE1) and 1.9mM theophylline added to the whole blood, platelet-rich plasma (PRP), and/or the storage medium during the preparation of platelet concentrates. In vitro markers of platelet aggregation, respiration, and cell integrity were measured over a 20-day storage period and evaluated in an analysis of variance. We found that a single-step addition of PGE1 and theophylline to the PRP prior to centrifugation was not sufficient in terms of preventing a rapid fall in pH, rise in pO2, fall in pCO2, loss of hypotonic shock response, and loss of aggregation response, compared to the addition of the inhibitors to the storage medium used to resuspend the platelet pellet. Factorial analysis showed that a reduction in the surface-to-volume ratio of the storage container further improved the maintenance of platelet respiration and, for three in vitro markers (hypotonic shock response, released lactic dehydrogenase, and surface glycoprotein Ib levels) displayed an interactive effect with the inhibitors. The addition of protease inhibitors to the formulation of PGE1 and theophylline showed further improvement in several markers. These findings demonstrate the possibility of preserving platelets for 15–20 days with the synergistic effects of activation inhibitors and an electrolyte storage medium fortified with citrate, buffers, and dextrose. In addition, these data suggest that platelet agonists generated in plasma accelerate the in vitro aging process of stored platelets.

The relationship between in vivo behavior and in vitro characteristics of 59 platelet concentrates (PC) stored for up to 14 days in a synthetic medium or in CPDA-1 plasma was systematically investigated. 25 paired studies (1 study was incomplete) were performed comparing platelets suspended either in the synthetic medium or CPDA-1 plasma with 5 days (n = 5); 7 days (n = 10); 10 days (n = 5); and 14 days (n = 5) of storage. In addition, 10 control studies were performed with freshly prepared PC (6–24 h) in CPDA-1 plasma. Both percent recovery and survival estimations showed decreases with increasing storage duration, irrespective of storage medium used. In both media, with prolonged storage, the platelet survival curves not only became shorter, but also increasingly exponential, suggesting that in vitro storage caused progressive damage to the platelets present in circulation. Survival curves of platelets suspended in synthetic medium remained more linear, indicative of less random damage during storage. Mean population lifespan (MPL) of the stored PC was determined by the area below the survival curve divided by the mean percent recovery for the fresh PC, which was 55%. MPL decreased from 4.5 days (fresh PC) to 0.4 days after 14 days of storage in plasma, with a 50% reduction (t½) estimated at 7.2 days of storage. MPL t½ for PC stored in the synthetic medium was estimated to be 8.8 days. The decrease in in vivo viability with prolonged storage was paralleled with loss of energy-dependent in vitro parameters such as hypotonic shock response, shape change with ADP, and ATP levels, and with increased lactate levels. Although the length of the storage period was shown to be the major factor responsible for the variability in MPL observed in this study, multiple linear-regression analysis showed also that platelet discoid shape, as measured by the shape change, and lactate production were independent in vitro parameters with significant predictability of the in vivo viability. Combined with days of storage in the regression equation, these parameters were found to ‘explain’ 78% of the variability of the MPL found in this study.

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. In all media there was a high correlation between platelet ATP level and the other in vitro parameters during storage. Furthermore, energy depletion of the platelets produced by using the physiologic salt solution without glucose and by addition of metabolic inhibitors caused a similar deterioration of in vitro functions, but at a significantly faster rate which correlated with the rate of decrease in platelet ATP. This suggests that the platelet lesion observed with prolonged storage in second-generation containers might be due to a failure in platelet energy metabolism.

Platelet concentrates collected by continuous flow automated apheresis (Fenwal CS-3000) were compared with those collected by manual apheresis to determine whether the prolonged centrifugation and vigorous resuspension affected platelet viability and in vitro function. Paired autologous reinfusion studies (111Indium) of 5 normal donors showed no significant differences in the mean percent recoveries (50.8 +/- 7.0% vs 53.8 +/- 4.0%) or survivals (174 +/- 23 h vs 188 +/- 10 h) for platelets collected by manual versus automated apheresis. Platelets collected by automated apheresis had a significantly higher level of beta-thromboglobulin release, but there were no significant differences between platelets collected by the two methods in regard to other in vitro parameters (ATP levels, LDH released, hypotonic shock response) believed to reflect platelet activation, injury, and malfunction. These results suggest that only slight activation of platelets takes place during automated apheresis.

Removal of leukocytes from platelet concentrates (PC) may decrease the incidence of alloimmunization to HLA antigens on white cells and prevent or delay refractoriness. Cotton wool filtration, which effectively removes leukocytes from PC with minimal platelet loss, may cause platelet or complement activation. In this study, the effect of cotton wool filtration (Imugard® IG-500) on platelet activation, in vitro function, posttransfusion survival, and complement activation was investigated. Five paired autologous in vivo percent recovery and survival studies were performed with fresh (6–8 h) PC or with 5-day-stored PC prepared from CPD-anticoagulated blood and stored in PL-732TM containers using standard methods. In the paired design, PC from the same donor were filtered on one occasion, and not filtered on another, prior to labeling with indium-111-oxine and reinfusion. Percent recovery and survival were then determined by the standardized method. Filtration had no statistically significant effect on percent recovery on PC stored for 6–8 h or for 5 days. There was, however, a slight decrease in survival hours of the filtered PC which was statistically significant at 6–8 h (203±14 vs. 179±18 h; p 0.05). Samples taken before and after filtration of single units (fresh and 5-day) and pooled units (3-day) for measurement of release of granular content (β-thromboglobulin), lysis (LDH), and in vitro viability—ATP, extent of shape change with ADP, and hypotonic shock response (3-day only)—demonstrated no effect of filtration on these parameters. A slight, but significant (p

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.

An in vivo and in vitro study of 42-day saline, adenine, glucose, mannitol (SAGM) red cells (Terumo Corporation, Elkton, Md.) was conducted using 20 volunteers to document in vivo efficacy and analyze the validity of the 99mTc/51Cr technique. In vivo autologous post-transfusion recovery was measured by a double-label procedure involving the use of 99mTc-labeled freshly drawn red cells to quantify recipient blood volume and 51Cr to document both post-transfusion 24-hour recovery (PTR) and red cell survival. As an internal control, 5 of the 20 donors studied had red cell volumes estimated by the 125I-albumin (RISA) plasma volume technique on a separate occasion. For comparison, PTR was also calculated according to the single-label 51Cr protocol in which recipient blood volume was estimated by extrapolation from circulating 51Cr-labeled red cell activity 5-15 min after infusion. After 42 days of storage, PTR averaged 78 +/- 6% with the double-label 99mTc/51Cr technique and 81 +/- 5% with the single-label (51Cr alone), confirming a small difference between the two techniques. Correlation between the two techniques was high, r = 0.81, though the average 3% difference between them was significant (p less than 0.05). Red cell volumes of the 5 donors measured by both the 99mTc-red cell method and the 125I-albumin method exhibited excellent correlation, r = 0.96, and averaged 1,961 +/- 420 ml and 2,048 +/- 381 ml, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)