Markers of platelet activation and apoptosis during storage of apheresis- and buffy coat-derived platelet concentrates for 7 days

BACKGROUND: The different production methods for platelet concentrates (PCs) result in products with variable in vitro quality and in vivo viability. The aim of this study was to compare in vitro variables of PCs produced by apheresis (AP-PC) or the buffy coat (BC-PC) method by applying a number of new and established assays.
STUDY DESIGN AND METHODS: Standard TRIMA Accel (Gambro BCT) AP-PCs (n = 20) and BC-PCs (n = 20) were stored in 100 percent plasma and changes in mitochondrial membrane potential (ΔΨ_m) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethlybenzimidazolcarbocyanine iodide (JC-1) dye on Days 1, 3, 5, and 7. The capacity of platelets (PLTs) for oxidative phosphorylation was also monitored by measuring oxygen consumption using a Clark-type electrode. PLT viability was measured using a new assay that utilizes the vital stains calcein-AM and FM4-64. Expression of phosphatidylserine (PS), CD42b, CD47, CD61, and CD62P was also assessed.
RESULTS: Although the JC-1 ratio (FL2/FL1) decreased significantly in both preparations, the percentage of PLTs with depolarized ΔΨ_m increased significantly in BC-PCs but not in AP-PCs. However, no significant change was detected in the PLTs' ability to consume oxygen in both preparations. PLTs in BC-PCs also showed significantly lower GPIb, CD47, and CD61 expression than AP-PCs on Day 1. PLTs in both preparations, however, showed a similar increase in CD62P and PS expression during storage, without significant loss of viability.
CONCLUSIONS: PLTs in AP-PCs and BC-PCs undergo different degrees of deterioration in mitochondrial integrity and thus may undergo different degrees of apoptosis. Interventions that maintain mitochondrial integrity may improve PLT viability in vitro.     

Platelet capacity of various platelet pooling systems for buffy coat-derived platelet concentrates

BACKGROUND: Platelet (PLT) storage lesions might depend on the total PLT count in the storage container and also on the PLT pooling system, especially the storage container, that is used for preparation of PLT concentrates (PCs). In this study, the PLT capacity of four commercially available PLT pooling systems was studied. MATERIALS AND METHODS: Four PCs were prepared in pooling systems of Baxter, Fresenius, Terumo, or Pall. The PCs were pooled and divided with various total PLT counts over the four storage containers (<225 × 10⁹, 225 × 10⁹‐324 × 10⁹, 325 × 10⁹‐424 × 10⁹, and >424 × 10⁹ PLTs). Volumes were kept equal by adding plasma to PCs with less than 425 × 10⁹ PLTs until a same volume as for PCs with more than 424 × 10⁹ PLTs was reached. PCs were stored at room temperature and tested for various in vitro variables on Days 1, 3, 5, 7, and 9. Paired experiments were repeated for each system five times. RESULTS: In vitro variables remained good for 9 days, that is, swirling score of 2 or more, pH value of 6.8 or more, glucose level of 10 mmol per L or more, lactate level of less than 25 mmol per L, and CD62p expression of less than 50 percent, for PCs in Baxter systems with more than 225 × 10⁹ PLTs, for PCs in Fresenius and Terumo systems with 225 × 10⁹ to 424 × 10⁹ PLTs, and for PCs in Pall systems with fewer than 425 × 10⁹ PLTs. CONCLUSION: PLT capacity depended on the PLT pooling systems used. All systems provide acceptable storage conditions. The Baxter system was the only system with capacity for more than 424 × 10⁹ PLTs per PC.     

Low cytokine contamination in buffy coat-derived platelet concentrates without filtration

BACKGROUND: Cytokines (interleukin [IL]-1 beta, IL-6, and tumor necrosis factor [TNF]) generated by white cells during the storage of platelet concentrates can cause febrile nonhemolytic transfusion reactions. The high rate of febrile reactions reported in other studies was not observed in the patients in the authors' center. This discrepancy prompted the determination of cytokine levels in buffy coat- derived platelet concentrates. STUDY DESIGN AND METHODS: Platelet concentrates were produced from buffy coats by a standard large-scale production process. Buffy coats were separated from the red cell and plasma components, and then platelets were recovered from the buffy coats by a soft-spin procedure. Levels of cytokines (IL-1 beta, IL-6, IL-8, and TNF) were determined with commercial enzyme-linked immunosorbent assays. RESULTS: In platelet concentrates produced by the buffy coat method, IL-1 beta, IL-6, IL-8, and TNF were observed at or below the detection limit of current enzyme-linked immunosorbent assays after 5 days' storage at 22 +/− 2 degrees C. Therefore, prestorage filtration had no measurable effect on cytokine levels. In controls, IL- 1 beta, IL-6, IL-8, and TNF were quantitatively detected after exogenous addition of recombinant cytokines or exposure to lipopolysaccharide. CONCLUSION: Platelet concentrates prepared from buffy coats may be virtually free of cytokines (IL-1 beta, IL-6, IL-8, and TNF) during 5 days of storage. Filtration is not required to reduce the recipient's cytokine exposure via such platelet concentrates.     

Overnight or fresh buffy coat-derived platelet concentrates prepared with various platelet pooling systems.

BACKGROUND: For logistic reasons, possibilities to produce both platelet (PLT) concentrates prepared from fresh or overnight-stored whole blood (fresh and o/n PCs, respectively) are convenient. The consequences of both possibilities are not well described. The PLT pooling system used might also influence the condition of PCs. Our aim was to compare fresh and o/n PCs with different PLT pooling systems. STUDY DESIGN AND METHODS: Fresh and o/n PCs were prepared from buffy coats and plasma in PLT pooling systems of Baxter, Fresenius, Terumo, or Pall (n = 5). PCs were stored for 9 days. The in vitro quality was determined by the PLT count, pH, glucose, lactate, pO(2), pCO(2), CD62P expression, and annexin V binding. RESULTS: The o/n PCs showed higher PLT count (approx. 460 x 10(9)/PC vs. approx. 310 x 10(9)/PC), pCO(2), and lactate concentration and lower pH, pO(2), glucose concentration, CD62P expression (until Day 5), and annexin V binding (until Day 7) compared with fresh PCs (p < 0.05). Only for o/n PCs in the Baxter and Fresenius systems did the pH and glucose concentration remain higher, and the lactate concentration and CD62P expression remained lower than that of o/n PCs in the Pall and Terumo systems. The pH for fresh PCs in the Baxter and Fresenius systems was more often greater than 7.4 than for fresh PCs in the Terumo or Pall systems. CONCLUSION: The quality of PCs depended on whether PCs were prepared from fresh or overnight-stored whole blood and on the used PLT pooling system. The main difference between fresh and o/n PCs was the PLT count.     

Freezing of buffy coat-derived, leukoreduced platelet concentrates in 6 percent dimethyl sulfoxide

BACKGROUND: There has recently been renewed interest in freezing platelets (PLTs) in dimethyl sulfoxide (DMSO) for the treatment of major traumatic injuries, especially in military situations. This study examined PLTs that were frozen in small volumes of 6 percent DMSO at ?80°C. STUDY DESIGN AND METHODS: Buffy coat–derived pooled leukoreduced PLT concentrates were frozen in 6 percent DMSO and stored at ?80°C. Assays included hypotonic shock response (HSR); aggregation; glycoprotein (GP)Ibα and P‐selectin binding sites; annexin V binding to phosphatidylserine, glycocalicin, and lactate dehydrogenase (LDH). Cone and plate technology (DiaMed Impact‐R, DiaMed) was used to test PLT function under near physiologic conditions. RESULTS: The freeze‐thaw loss of PLTs was 23 percent. HSR was 17 ± 7 percent. Cytometry demonstrated two populations of PLTs: one with normal levels of GPIbα binding sites (27 × 10³ ± 3 × 10³/PLT) and one with reduced levels (5.5 × 10³ ± 1.2 × 10³/PLT). There were 1.4 × 10³ ± 0.2 × 10³ P‐selectin binding sites per PLT. Annexin V binding to phosphatidylserine was 50 ± 9 percent and LDH was 496 ± 207 IU per 10¹² PLTs. Surface coverage and aggregate size, as measured by the DiaMed Impact‐R, were similar to those observed with PLTs stored for 2 days at 22°C. CONCLUSION: Some degree of activation was demonstrated by the proportion of PLTs with reduced levels of GPIbα binding sites, increased P‐selectin expression, and increased Annexin V binding. LDH concentrations indicated a degree of lysis. The DiaMed Impact‐R results showed that the PLTs were still capable of adhering to surfaces and forming aggregates under shear force.     

In-process cell counts during preparation of platelet concentrates from buffy coats

To ensure the quality of platelet concentrates (PCs), we studied in-process recoveries of blood cell counts in pooled PCs derived from four or five buffy coats (BCs) from Biopack Compoflex Systems in Bern (PC-BC/4 or PC-BC/5) and from five BCs from Optipac (Baxter) in Zurich (PC-BC/5). BCs were pooled employing a sterile connecting device and flushing them with 300 mL of platelet additive solution. The pools were centrifuged for 12 min at 500 g at 20 degrees C and filtered with PALL's Auto-Stop BC-leukocyte removal filter. Automated platelet counting was performed on whole blood donation, on single BC, on pooled BC and in the final product. Four out of 10 PC-BC/4 (= 40%) and 29 out of 30 PC-BC/5 (= 97%) had a total platelet count of > 200 x 10(9) platelets. Average percentage recoveries in PC compared to the pre-centrifugation BC pools were similar with the Biopack Compoflex and the Optipac systems, 62% and 57% respectively, whereby the absolute platelet count per one donation was similar, i.e. 49.5 x 10(9), 55 x 10(9) and 53 x 10(9) in PC-BC/4 and PC-BC/5 from Bern and PC-BC/5 from Zurich. There was a significant positive correlation between the inital number of BCs taken for pooling and the final platelet counts in the PCs. In order to recover a minimal platelet content of 200 x 10(9) platelets per pooled unit, it is safer to start out with five rather than with four donations unless recoveries during the production steps can be improved.     

Platelet storage lesion of WBC-reduced, pooled, buffy coat-derived platelet concentrates prepared in three in-process filter/storage bag combinations

BACKGROUND: With the implementation of universal WBC reduction in the United Kingdom, in-process WBC-reduction filters for pooled buffy coat (BC)-derived platelet concentrates (PCs) are used in routine production. The effects of three filter/storage bag combinations on platelet activation and microvesiculation and on the activation of coagulation were investigated. STUDY DESIGN AND METHODS: Using pooled BCs from the same donors, three filter/storage bag combinations (Autostop BC/CLX, Pall Biomedical; Sepacell PLX5/PL2410, Asahi Medical; and Imugard III-PL 4P/Teruflex, Terumo) were compared with unfiltered controls for their effects on microvesiculation and other storage-induced changes in platelets. Process efficiency was measured by platelet yield and residual WBC count. The storage changes were assessed: pH, activation of platelets measured by CD62P on the platelet surface and in supernatant plasma, quantitation of platelet-derived and RBC-derived microvesicles, cellular injury measured by annexin V in the supernatant plasma, and activation of the coagulation system measured by kallikrein-like and thrombin-like activities, prothrombin fragment 1+2, and thrombin-antithrombin complex. RESULTS: All three filters were comparable in terms of platelet recovery and WBC removal, and none induced immediate platelet activation or microvesiculation. With storage, platelet activation or microvesiculation increased in platelets prepared by all three filters and in unfiltered controls, but these effects were significantly less in the Imugard PCs than in controls. These findings were consistent with those for annexin V in the supernatant plasma, which were lower in Imugard PCs than in other products. Sepacell and Imugard filters reduced RBC-derived microvesicles to 50 percent of control levels, but the Autostop filter had no effect. On storage, levels of RBC-derived microvesicles in filtered products remained static, but levels in the unfiltered control doubled. Kallikrein- and thrombin-like activities were generated only by the Autostop filter without any further increment on storage. CONCLUSION: WBC-reduced pooled BC-PCs prepared by various filter/bag combinations were equivalent on Day 1 but differed during storage in terms of platelet activation or microvesiculation.     

白膜放置时间对制备手工浓缩血小板质量的影响

目的评价全血当天分离的白膜(BC)在不同时间分离制备的浓缩血小板(PC)的质量,为手工制备PC提供参考。方法将80袋400 mL全血于采集6 h内分离出BC,将5袋同血型BC由无菌接驳机对接合并成1袋(1个治疗量)后,再均分在3个血小板保存袋内,1袋即刻(0 h组)轻离心分离制备PC,另2袋在22℃血小板保存箱分别振摇4 h(4 h组)和16 h(16 h组)后再分离PC。对所有标本留样进行血小板质量检测,包括Plt、血小板回收率、CD62P、聚集率、RBC混入量、WBC混入量、FHb含量。结果 3组PC制剂RBC混入量、Plt、血小板回收率、CD62P、聚集率差异无统计学意义;WBC混入量:0 h(6.76±1.29)和4 h组变化不明显,16 h(3.78±0.45)组降低明显(P<0.05);FHb含量:随BC处理时间延长有增高趋势,16 h(65.62±11.11)与0 h(33.45±6.95)比差异具统计学意义(P<0.05)。结论随BC放置时间延长对制备PC制剂的质量有一定影响。     

Generation of kinins during preparation and storage of whole blood-derived platelet concentrates

BACKGROUND: Leukoreduction of platelet (PLT) concentrates (PCs) may be associated with hypotension in recipients, and a role for bradykinin (BK)-related peptides has been proposed for this side effect. STUDY DESIGN AND METHODS: The concentration of BK and one of its vasoactive metabolites, des-arginine(9)-BK (des-Arg(9)-BK), was measured in a large number of PCs as a function of leukoreduction and storage duration with specific enzyme immunoassays and complementary techniques. RESULTS: On Day 0 of storage, kinins were detected in leukoreduced and unfiltered PCs at a concentration lower than 100 pg per mL. During storage, both kinin levels peaked on Day 5 of storage, with a concentration higher than 1 ng per mL in 22 percent of PCs whether filtered on Day 0 or not. Physicochemical and pharmacologic characterizations of immunoreactive kinins confirm their nature. In vitro activation of the contact system of the corresponding PLT-poor plasma showed that a high kinin concentration on Day 5 of the storage corresponded with a low kinin-forming capacity of plasma. On Day 7, BK was no longer elevated presumably due to its degradation and the depletion of kinin-forming capacity of the plasma in stored PCs. The activities of metallopeptidases that metabolize BK-related peptides in plasma from PCs were at levels similar to those recorded in the plasma of a normal reference population and were unaffected by storage. CONCLUSION: Storage of PCs contributes to the hydrolysis of high-molecular-weight kininogen and generation of pharmacologically relevant BK levels that might pose a hazard in susceptible patients.     

Platelet membrane glycoprotein changes during the preparation and storage of platelet concentrates

Previous studies of platelet membrane glycoproteins during blood bank storage have reported conflicting results. This study assessed two major plasma membrane glycoproteins (GP Ib and GP IIb), an alpha-granule membrane protein (GMP-140), and the concentration of platelet membrane microparticles in cell-free plasma during routine hospital blood bank platelet storage. 125I-monoclonal antibody binding was used to measure membrane glycoproteins on the surface of intact platelets and to measure the concentration of membrane microparticles in cell-free plasma. Platelet concentrates were stored at room temperature in polyolefin bags for 7 days. In this blood bank, two types of rotators are routinely used for platelet concentrate storage: a 2-rpm circular tumbler rotator and a 6-rpm elliptical rotator. Different results were obtained with the rotators. With the tumbler rotator, there was no loss of platelets and antibody binding to GP Ib remained normal. With the elliptical rotator, one third of platelets were lost into clumps during storage, and a 50 percent decrease of antibody binding to GP Ib occurred in the remaining single platelets. There was no loss of antibody binding to GP IIb with either rotator. Antibody binding to GMP-140 increased equally in both rotators indicating that the remaining single platelets had secreted about 16 percent of their alpha-granule contents. The plasma concentration of platelet membrane microparticles was greater in the bags stored in the elliptical rotator. These results indicate that it is possible to maintain the normal concentration of platelet membrane glycoproteins Ib and IIb during 7 days of room-temperature blood bank storage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Superior integrin activating capacity and higher adhesion to fibrinogen matrix in buffy coat-derived platelet concentrates (PCs) compared to PRP-PCs

Background

Regardless of different sources, methods or devices which are applied for preparation of therapeutic platelets, these products are generally isolated from whole blood by the sedimentation techniques which are based on PRP or buffy coat (BC) separation. As a general fact, platelet preparation and storage are also associated with some deleterious changes that known as platelet storage lesion (PSL). Although these alternations in platelet functional activity are aggravated during storage, whether technical issues within preparation can affect integrin activation and platelet adhesion to fibrinogen were investigated in this study.

Monitoring of platelet morphology during storage of platelet concentrates

During storage, human platelet concentrates progressively lose the capacity to survive and function in vivo after transfusion. A shape transformation from disc to sphere is the most reliable in vitro determinant for the loss of the in vivo survival of platelets. To find an objective measurement for platelet morphology, we studied the effect of anticoagulant, temperature, and storage on the apparent median platelet volume (MPV) as determined by a particle counter and on changes in platelet shape as measured and by light microscopy. Changes in MPV, light transmission, and morphology score by light microscopy were observed within 1 minute after collection of blood in CPDA. As compared to blood immediately fixed on withdrawal, in CPDA blood, the MPV increased from 4.1 to 5.7 fl, and light transmission difference decreased from 22 to 7 percent. A partial restoration of these determinants was found when the whole blood was incubated for 30 minutes at 37 degrees C, before preparation of platelet-rich plasma. In the first 5 days of platelet storage, the MPV increased from 4.6 to 5.0 fl; thereafter, it started to decrease. An increase in fragmented platelets after 5 days was observed on light microscopy. The light transmission difference showed a slow disc-to-sphere transformation during storage. This transformation accelerated from Day 5 to Day 7; after 11 days, only spheres were detected. After 7 days the swirling pattern scores were still in accordance with the presence of discs, whereas the other structure-associated determinants showed already spheric and even fragmented platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship of the time of storage and transfusion reactions to platelet concentrates from buffy coats

BACKGROUND: Transfusion reactions to platelet concentrates prepared from buffy coats (BC-PCs) were reviewed to determine the effect of some variables of BC-PC preparation and storage: time of BC storage before BC-PC preparation (1-2 days); time of BC-PC storage before transfusion (1-5 days); no white cell reduction versus laboratory and bedside BC-PC white cell reduction. STUDY DESIGN AND METHODS: A multiple linear logistic regression model was used by which the relative effect of one variable is expressed as the relative risk of transfusion reaction against a baseline level (1-day storage, no white cell reduction). RESULTS: During the 14 months of study, a total of 2707 BC-PC transfusions were given to 192 patients; 37 reactions (1.4%) were reported in 25 patients (13%). The transfusion reactions were febrile, nonhemolytic in 23 cases; allergic in 5; febrile and allergic in 2; and other in 7. The relative risk of transfusion reaction to BC-PCs prepared from BCs stored for 2 days was 1.98 times that to BC-PCs prepared from BCs stored for 1 day (p = 0.07). The relative risk of transfusion reaction of 5-day-old BC-PCs was 10.7 times that of 1-day- old BC-PCs (p = 0.001). The relative risk of transfusion reactions of BC-PCs white cell-reduced in the laboratory and at the bedside were 0.65 (p = 0.3) and 1.87 (p = 0.1) times, respectively, that of non- white cell-reduced BC-PCs. CONCLUSION: Time of storage seems to be an important variable associated with BC-PC transfusion reaction.     

Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods

The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from platelet-rich plasma (PRP) and from the other as nonpelleted platelets from the buffy coat (BC). The activation of platelets in these PCs was studied immediately after preparation and during storage for up to 9 days at 22 degrees C with gentle agitation. The binding of monoclonal antibodies (MoAbs) against the GP IIb/IIIa complex and against activation-dependent antigens (GMP 140 from the alpha granules and a 53-kDa glycoprotein from the lysosomal granules) was measured. Beta-thromboglobulin (beta-TG) release was also determined. Disc-to-sphere transformation was quantitated by measuring on an aggregometer the difference in light transmission during stirring at different rates and also by light microscopy. Immediately after preparation, platelets derived from PRP had a more spheric morphology (p less than 0.01), had a higher beta-TG release (p less than 0.01), bound more MoAbs against GP IIb/IIIa (p less than 0.01), and expressed more GMP 140 and 53-kDa glycoprotein (p less than 0.01) than did BC-derived platelets. However, these differences had disappeared after 2 days of storage. It was concluded that, immediately after preparation, PRP-derived platelets are more activated than BC-derived platelets. This is most likely a result of the pelleting that follows the second high-speed centrifugation of the PRP.     

Ammonia accumulation in platelet concentrates during storage

Newly prepared platelet concentrates (PC) and platelet poor plasma (PPP) stored at either room temperature (RT) or 4 C were analyzed for ammonia content initially, and after storage at 24, 48, and 72 hours. These studies demonstrate that ammonia rapidly accumulates in PC stored at RT. This accumulation appears to be temperature dependent, as evidenced by the observed differences in ammonia accumulation in PC stored at 4 C and RT. Whether ammonia acts as a toxic metabolite in the inhibition of platelet function and the development of the "storage lesion," which occurs in PC stored at RT, needs further clarification. Additional studies are also necessary to establish the clinical efficacy of transfusing ammonia-containing platelet concentrates into premature neonates with immature functioning livers, and perhaps into adults with severe liver disease.