Liquid-stored Platelets Research Articles

A randomized cross-over study of cryopreserved platelets in prophylactic transfusions of thrombocytopenic patients.

The short shelf-life of liquid-stored platelets (LP) at 20-24°C poses shortage and wastage challenges. Cryopreserved platelets have significantly extended shelf-life, and were safe and efficacious for therapeutic transfusions of bleeding patients in the Afghanistan conflict and phase 2 randomized studies. Although hematology patients account for half of platelets demand, there is no randomized study on prophylactic cryopreserved platelet transfusions in them. We performed a phase 1b/2a randomized cross-over study comparing the safety and efficacy of cryopreserved buffy coat-derived pooled platelets (CP) to LP in the prophylactic transfusions of thrombocytopenic hematology patients. A total of 18 adults were randomly assigned 1:1 to CP and LP for their first thrombocytopenic period (TP) of up to 28-days. A total of 14 crossed over to the other platelet-arm for the second TP. Overall, 17 subjects received 51 CP and 15 received 52 LP. CP-arm had more treatment emergent adverse event (29.4% vs. 13.3% of subjects, 9.8% vs. 3.8% of transfusions) than LP-arm but all were mild. No thromboembolism was observed. Both arms had similar bleeding rates (23.5% vs. 26.7% of subjects) which were all mild. Subjects in CP-arm had lower average corrected count increments than LP-arm (mean [SD] 5.6 [4.20] vs. 22.6 [9.68] ×109 /L at 1-4 h, p < .001; 5.3 [4.84] vs. 18.2 [9.52] ×109 /L at 18-30 h, p < .001). All TEG parameters at 1-4 h and maximum amplitude (MA) at 18-30 h improved from baseline post-CP transfusion (p < .05) though improvements in K-time and MA were lower than LP (p < .05). During shortages, CP may supplement LP in prophylactic transfusions of thrombocytopenic patients.

Cryopreserved platelets compared with liquid-stored platelets for the treatment of surgical bleeding: protocol for two multicentre randomised controlled blinded non-inferiority trials (the CLIP-II and CLIPNZ-II trials)

IntroductionCryopreservation at −80°C in dimethylsulphoxide extends platelet shelf-life from 7 days to 2 years. Only limited comparative trial data supports the safety and effectiveness of cryopreserved platelets as a treatment...

Design of the Randomized Controlled Trial Comparing Dimethyl Sulfoxide Cryopreserved Platelets to Liquid Stored Platelets in Patients Undergoing Cardiopulmonary Bypass Surgery

ObjectiveDimethylsulfoxide-cryopreserved platelets are being evaluated for treatment of acute hemorrhage in patients with thrombocytopenia or platelet dysfunction when liquid stored platelets are unavailable. Patients undergoing cardiac surgery with cardiopulmonary bypass with risk factors for significant bleeding represent a population for which determining efficacy and safety of cryopreserved platelets is ideal in the clinical trial setting. The primary objective is to compare blood loss in cardiopulmonary bypass patients receiving cryopreserved platelets or liquid stored platelets. MethodsIn patients undergoing cardiac surgery utilizing cardiopulmonary bypass, a standardized algorithm with transfusion triggers will be used to guide the intra- and postoperative administration of study platelets, either cryopreserved platelets or liquid stored platelets, based on the clinical presentation. The primary efficacy end point was the volume of blood loss from completion of chest closure (time 0) until the time chest tubes were removed or 24 hours after chest closure, whichever is earlier. ResultsThis design article describes an ongoing multicenter, randomized, blinded trial to evaluate noninferiority or superiority of cryopreserved platelets with liquid stored platelets in controlling blood loss in patients undergoing cardiopulmonary bypass surgery. ConclusionsFrozen storage could substantially safely extend the shelf life of stored platelets. If efficacy and safety were demonstrated in this trial, current constraints on platelet use in low resource military and civilian settings would be relieved.

A pilot randomized clinical trial of cryopreserved versus liquid-stored platelet transfusion for bleeding in cardiac surgery: The cryopreserved versus liquid platelet-New Zealand pilot trial.

Platelets for transfusion have a shelf-life of 7 days, limiting availability and leading to wastage. Cryopreservation at -80°C extends shelf-life to at least 1 year, but safety and effectiveness are uncertain. This single centre blinded pilot trial enrolled adult cardiac surgery patients who were at high risk of platelet transfusion. If treating clinicians determined platelet transfusion was required, up to three units of either cryopreserved or liquid-stored platelets intraoperatively or during intensive care unit admission were administered. The primary outcome was protocol safety and feasibility. Over 13 months, 89 patients were randomized, 23 (25.8%) of whom received a platelet transfusion. There were no differences in median blood loss up to 48 h between study groups, or in the quantities of study platelets or other blood components transfused. The median platelet concentration on the day after surgery was lower in the cryopreserved platelet group (122 × 103 /μl vs. 157 × 103 /μl, median difference 39.5×103 /μl, p= 0.03). There were no differences in any of the recorded safety outcomes, and no adverse events were reported on any patient. Multivariable adjustment for imbalances in baseline patient characteristics did not find study group to be a predictor of 24-h blood loss, red cell transfusion or a composite bleeding outcome. This pilot randomized controlled trial demonstrated the feasibility of the protocol and adds to accumulating data supporting the safety of this intervention. Given the clear advantage of prolonged shelf-life, particularly for regional hospitals in New Zealand, a definitive non-inferiority phase III trial is warranted.

Reconstituted cryopreserved platelets synthesize proteins during short-term storage and packaging a defined subset into microvesicles.

Cryopreservation of platelets (PLTs) could allow extension of their shelf-life to years, compared to days for liquid stored platelets. Due to their greater hemostatic effect, reconstituted cryopreserved platelets (cryo-PLTs) would be able to support bleeding emergencies. Since protein synthesis has been linked to PLT functions, such as clot formation and immune responses, the translational capacity of reconstituted cryo-PLTs was assessed upon thawing and short-term storage. Platelets were frozen at -80°C with 5-6% DMSO. Upon thawing, they were reconstituted in plasma and then aliquoted (12 ml) into mini-bags and assessed over 24 h of storage at RT. One series served as control; the second and third series were spiked with either 300 μM puromycin (Pm) or 227 nM biotin-labeled Pm. Samples were tested for in vitro quality and PLT microvesicle enumeration by flow cytometry. Protein synthesis in cryo-PLTs was assessed using a modified method based on puromycin-associated nascent chain proteomics. In vitro parameters of reconstituted and subsequently stored platelets were consistent with previously published results. Mass-spectrometry analyses identified that 22 proteins were synthesized in PLTs and 13 of those were observed in platelet microvesicles (PMVs). Cryo-PLTs can synthesize proteins upon reconstitution and storage. Discovery of a subset of these proteins in the PMV suggests a role in vesicle encapsulation, possibly in a selective manner. This observation provides novel insights into the capacity for protein synthesis in cryo-PLTs and the potential regulation of protein packaging into PMV.

Cryopreservation of buffy coat derived platelets: Paired in vitro characterization using uncontrolled versus controlled freezing rate protocols.

BackgroundCryopreserved platelets show a reduced recovery and viability after freezing and thawing including several ultrastructural and phenotypic deteriorations compared with liquid‐stored platelets. It is suggested that using Controlled‐Rate Freezing (CRF) can reduce variability and optimize the functionality profile for cells. The objective of the study is to compare cellular, metabolic, phenotypic and functional effects on platelets after cryopreservation using different freezing rate protocols.Study Design and MethodsTo evaluate the possible effects of different freezing rate protocols a two‐experimental study comparing diverse combinations was tested with a pool and split design. Uncontrolled freezing of platelets in materials with different thermal conductivity (metal vs cardboard) was evaluated in experiment 1. Experiment 2 evaluated uncontrolled vs a controlled‐rate freezing protocol in metal boxes. All variables were assessed pre and post cryopreservation.ResultsDirectly after thawing, no major differences in platelet recovery, LDH, ATP, Δψ, CD62P, CD42b, platelet endothelial cell adhesion molecule and sCD40L were seen between units frozen with different thermal conductivity for temperature. In contrast, we observed signs of increased activation after freezing using the CRF protocol, reflected by increased cell surface expression of CD62P, PAC‐1 binding and increased concentration of LDH. Agonist induced expression of a conformational epitope on the GPIIb/IIIa complex and contribution to blood coagulation in an experimental rotational thromboelastometry setup were not statistically different between the groups.ConclusionThe use of a uncontrolled freezing rate protocol is feasible, creating a platelet product comparable to using a controlled rate freezing equipment during cryopreservation of platelets.

Frozen Platelets-Development and Future Directions.

Storage requirements and outdating of platelets represent a continued challenge for blood banks. These hurdles are confounded for rural area hospitals or in military deployments. Over 60 years of research and development into frozen platelets have generated a stable and reproducible product. Valeri's method to freeze platelets in 6% dimethyl sulfoxide (DMSO) and storage at -80°C allows for long-term storage alleviating burdens placed on blood banks. Clinical studies show that frozen platelet transfusions are safe with no related thrombotic or other serious adverse events. There are ongoing efforts to demonstrate cryopreserved platelet (CPP) superiority in efficacy studies designed in trauma or cardiac surgery patients. Technical advances in CPP manufacturing including closed system manufacturing, applications of pathogen reduction technology and potency standard characterization add to the appeal of CPP as an alternative to traditional liquid-stored platelets (LP) in settings of supply shortages, mass casualty, active bleeding, rapid provision of HLA-compatible platelets, and remote care.

The use of cryopreserved platelets in a trauma-induced hemorrhage model.

BackgroundCryopreserved platelet products can be stored for years and are mainly used in military settings. Following thawing, cryopreserved platelets are activated, resulting in faster clot formation but reduced aggregation in vitro, rendering their efficacy in bleeding unknown. Also, concerns remain on the safety of these products. The aim was to investigate the efficacy and safety of cryopreserved platelets in a rat model of traumatic hemorrhage.Study Design and MethodsAfter 1 hour of shock, rats (n = 13/group) were randomized to receive a balanced transfusion pack (1:1:1 red blood cell:plasma:platelet) made from syngeneic rat blood, containing either liquid stored platelets or cryopreserved platelets. Primary outcome was the transfusion volume required to obtain a mean arterial pressure (MAP) of 60 mmHg. Secondary outcomes were coagulation as assessed by thromboelastometry (ROTEM®) and organ failure as assessed by biochemistry and histopathology.ResultsThe transfusion volume to obtain a MAP of 60 mmHg was lower in animals receiving cryopreserved platelets (5.4 [4.1‐7.1] mL/kg) compared to those receiving liquid stored platelets (7.5 [6.4‐8.5] mL/kg, p < 0.05). ROTEM® clotting times were shorter (45 [41‐48] vs. 49 [45‐53]sec, p < 0.05), while maximum clot firmness was slightly lower (68 [67‐68] vs. 69 [69‐71]mm, p < 0.01). Organ failure was similar in both groups.ConclusionsUse of cryopreserved platelets required less transfusion volume to reach a targeted MAP compared to liquid stored platelets, while organ injury was similar. These results provide a rationale for clinical trials with cryopreserved platelets in (traumatic) bleeding.

Assays for phenotypic and functional characterization of cryopreserved platelets

Platelet concentrates for transfusion have a limited shelf-life, and cryopreservation offers a means of extending platelet shelf-life for at least 2 years. Cryopreservation, however, has some disadvantages, as platelets can be damaged during the freezing and thawing process. Consequently the phenotype of cryopreserved platelets is very different to that of freshly collected, liquid stored platelets. To obtain a reliable overview of cryopreserved platelet quality and function, specific testing methodologies should be considered.Whilst light transmission aggregometry (LTA) is the gold standard for measuring in vitro platelet reactivity in liquid stored platelets, it is not suited for cryopreserved platelets, as they retain little response to typical platelet agonists, even at high doses. Instead, phenotypic characterization by flow cytometry in combination with global assays of coagulation provides a clearer delineation of the phenotype and function of cryopreserved platelets. With increasing international uptake of platelet cryopreservation, it is important to recognize the need for appropriate measures of platelet quality and function.

Transfusion of cryopreserved platelets exacerbates inflammatory liver and lung injury in a mice model of hemorrhage.

Platelets are essential for primary hemostasis and also play an important role in inflammatory reactions. The hemostatic property of cryopreserved platelets (CPPs) has been confirmed in the treatment of bleeding casualties, but inflammatory injury induced by CPP transfusion is relatively unclear. We aim to investigate the effects of CPP transfusion on inflammatory organ injury in mice after hemorrhage. Mice were subjected to a volume-controlled hemorrhage over 1 hour, and then were transfused with fresh platelets (FPs), Liquid-stored platelets (LPPs), CPPs, or fresh frozen plasma (FFP, control). At 6 hours posttransfusion, mice were sacrificed, and blood and tissues were sampled. Tissue sections were examined histologically and by immunohistochemical staining of neutrophils and macrophages. Plasma alanine aminotransferase, hepatic myeloperoxidase activity and inflammatory cytokine levels were measured. Transfusion of stored platelets (LPPs and CPPs) caused more serious histological injury in liver and lung compared with FPs and FFP (p < 0.05). However, kidney histological injury was similar among groups. Significantly higher numbers of Ly-6G-positive neutrophils were detected in liver and of F4/80-positive macrophages in liver and lung of mice transfused with LPPs or CPPs compared with FPs or FFP (p < 0.05). Transfusion of CPPs caused the most severe inflammatory liver injury, as reflected by alanine aminotransferase levels, hepatic macrophage infiltration, and hepatic myeloperoxidase activity and inflammatory cytokine levels (macrophage inflammatory protein-2, tumor necrosis factor-α, and interleukin-1β). Cryopreserved platelet transfusion is more likely to aggravate hemorrhage-induced liver and lung injury by activating macrophage and facilitating neutrophil infiltration into hepatic tissues.

Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets

BackgroundFreezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA) of CPPs.MethodsCPPs and corresponding liquid-stored PLTs (LSPs) were characterized by flow cytometry (FC), fluorescence polarization (FP), nanoparticle tracking analysis (NTA), electron microscopy (SEM, TEM), atomic force microscopy (AFM) and thrombin-generation (TG) test.ResultsSEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing–thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a+ PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent.ConclusionsMajor portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing–thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have significant contribution to PCA of CPPs.

Cryopreserved Platelets: From in Vitro Thrombin Generation Potential to in Vivo Safety

Background: Cryopreservation of platelets (PLTs) at -80°C with dimethyl sulfoxide (DMSO) can extend their shelf life up to 2 years. Cryopreserved PLTs (CRY-PLTs) are reported to have a greater in vivo hemostatic effect than liquid-stored PLTs. Aims of this study were: i. to evaluate the thrombin generation potential of buffy coat derived cryopreserved PLTs (CRY- BC PLT) in comparison with fresh buffy coat derived platelets concentrates; ii. to determine the efficacy and safety of CRY-PLTs transfusion in hematological patients with severe thrombocytopenia.Materials and methods: BC PLTs were obtained from 5 buffy coats and pooled. The final PLTs concentrates were leukoreduced by filtration and transferred to a 650 mL patented cryopreservation kit (Promedical ®) which allowed mixing with DMSO 25% in a closed system and following removal of supernatant without further manipulations. BC-PLTs were washed prior freezing, suspended in homologous plasma from 1 of the 5 donors to a final concentration of 200 mL and frozen at - 80°. CRY- BC PLTs were preserved at -80°C with 6% DMSO. A system of 3 accessory bags directly connected to the mother bag was adopted for the in vitro study, to avoid repeated freezing/thawing of samples. In vitro assays were performed before freezing and at 3,6 and 9 months after thawing. Before assay, CRY-BC PLT were thawed at 37°C and diluted in plasma to adjust to 300× 109/L PLTs. Fresh BC PLTs underwent the same dilution to adjust to 300 ×109/L PLTs. Thrombin generation (TGA) was tested in CRY BC-PLTs and compared to TG potential of fresh BC PLTs. TGA was triggered by the addition of 0.5 pmol/L of recombinant human tissue factor. Endogenous thrombin potential (ETP) and peak height (PH) were determined. Flow Cytometry assays for PLTs activation markers and thromboelastography were also determined on each sample.CRY-BC PLTs, separately prepared according to the above described method for in vivo study, were infused in five hematological patients with acute leukemia (AL) and severe thrombocytopenia (PLTs <10 ×109/L) participating to the trial NCT02032134.CRY-BC PLTs were transfused to control epistaxis (n=2) and for prophylaxis (n=3). Patients were observed up to 7 days after infusion and the occurrence of any side effect was registered. An increase in PLTs count was observed only in one case, under prophylaxis, but bleeding was successfully controlled or prevented in all cases. Plasma from patients transfused with CRY-BC PLTs was tested for TGA pre-treatment and 24 hours after treatmentResults Fourty nine BC-PLTs from 245 healthy volunteer donors (145 males and 100 females, mean age: 48.16.±18.91) were prepared, cryopreserved and analyzed up to 9 months after storage. Cryopreserved PLTs show a good thrombin generation potential that is stably maintained up to 9 months after cryopreservation [ETP (nM min): 529.25±98.64 at T0, 558.82±114.67 at T3, at 548.57±93.38 T6 and 533.04±103.15 at T9 months, respectively; PH(nM): 132.77±44.9 at T0, 103.4±44.9 at T3, 108.0±36.7 at T6 and 132.0±44.6 at T9 months, respectively]. At TGA, fresh BC-PLTs (n=35) had a mean ETP of 760.13±130.11, PH was 138.9±40.2. Thrombin generation of CRY-BC PLTs is comparable to fresh BC-PLTs, even if slightly decreased. Infusion of CRY-BC PLT (1U) was effective in controlling mucosal bleeding (epistaxis) in two patients with AL and severe thrombocytopenia. CRY-PLT were also effective when administered for prophylaxis in 3 patients with very low platelets count secondary to chemotherapy. In vivo, thrombin generation is stably maintained up to 24 hours after infusion of 1 Unit of CRY-BC PLTs, without any adverse effect (mean ETP pre-treatment was: 414.13±160.60, 24 hours after transfusion: 326.95±152.54). CRY-BC PLTs were safe and they did not determine any thrombotic event.At flow-cytometry, CRY-BC PLTs expressed higher activation markers (CD62P,CD63) than fresh BC PLTs. CRY-BC PLTs are able to significantly decrease the time to clot formation and clot strength, as measured also by thromboelastography. CRY-BCPLTs activation/deterioration is accompanied by an effective hemostatic in vivo function.Conclusions: Cryopreserved PLTs have an enhanced hemostatic activity and a good thrombin generation potential. They are effective and safe in preventing and controlling bleeding, being available in emergency/urgency settings also for patients with acute leukemia and severe thrombocytopenia. DisclosuresReina:Promedical: Consultancy.

The hemostatic activity of cryopreserved platelets is mediated by phosphatidylserine‐expressing platelets and platelet microparticles

Cryopreservation of platelets (PLTs) at -80°C with dimethyl sulfoxide (DMSO) can extend the shelf life from 5 days to 2 years. Cryopreserved PLTs are reported to have a greater in vivo hemostatic effect than liquid-stored PLTs. As such, the aim of this study was to understand the mechanisms responsible for the hemostatic potential of cryopreserved PLTs and the contribution of the reconstitution solution to this activity. DMSO (5% final concentration) was added to buffy coat-derived PLTs, followed by prefreeze removal of DMSO and storage at -80°C. Cryopreserved PLTs (n=8 per group) were thawed at 37°C, reconstituted with either 1 unit of thawed frozen plasma or PLT additive solution (PAS-G). In vitro assays were performed before freezing and after thawing to assess the hemostatic activity of PLTs. Cryopreserved PLTs expressed high levels of phosphatidylserine and contained significantly more phosphatidylserine-positive PLT microparticles than liquid-stored PLTs. This was accompanied by a significant decrease in the time to clot formation and clot strength, as measured by thromboelastography. The supernatant from cryopreserved PLTs was sufficient to reduce the phosphatidylserine-dependent clotting time and increase the thrombin generation potential. Overall, plasma-reconstituted cryopreserved PLTs were more procoagulant than those reconstituted in PAS-G. PLT cryopreservation results in the generation of phosphatidylserine-expressing PLT microparticles which contribute to the hemostatic activity. Understanding the hemostatic activity of these components may assist in extending the use of these specialized components beyond military applications.

Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution

Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me 2SO) and stored for extended periods at −80 °C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me 2SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at −80 °C. The cryopreserved platelet units ( n = 9) were rapidly thawed at 37 °C, reconstituted in 50% SSP+/plasma and stored at 22 °C. Platelet recovery and quality were examined 1 and 24 h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24 h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me 2SO and additive solution produces acceptable in vitro platelet quality.

Bacterial contamination in blood components and preventative strategies: an overview

Bacterial contamination of blood and its cellular components remains an unresolved problem in transfusion medicine and is considered to be the most common microbiological cause of transfusion associated morbidity and mortality. This is because contaminated units may contain large numbers of virulent bacteria as well as endotoxins that are considered to be fatal to the recipient. Accordingly, measures have been proposed to prevent or at least control the potential risk of transfusion associated bacteria infections. Broadly, these approaches include: bacterial avoidance; bacterial growth inhibition and bacterial load reduction by leucofiltration/viral inactivation. Unfortunately, none of the current approaches alone or in combination have received overall acceptance in terms of operational practice and safety/efficacy. Considerable effort has also been directed towards improving bacterial detection in order to provide a scientific basis for the lengthening of the shelf life of liquid stored platelets, without affecting, to a large extent their safety/efficacy. These issues have been highlighted in this overview on the current status and future trends.

L-carnitine and its possible role in red cell and platelet storage

The storage of red cells or platelets in the liquid state results in changes in quality over time. These changes are collectively known as the storage lesion and are associated with decreased in vivo viability and functionality. Modification of the components of the liquid milieu could favorably influence these changes. L-carnitine is a naturally occurring compound, which is best known for its role in facilitating the transport of long chain fatty acids across the mitochondrial membrane. An additional role for this compound may also exist as a reservoir of acylcarnitines to replace oxidized fatty acids in membrane phospholipids. In experimental studies, L-carnitine has been shown to reduce hemolysis and to improve in vivo survival in nonleukoreduced red cells. This effect on hemolysis appears to be attenuated by prestorage leukoreduction, but the practical benefit of this effect to transfusion recipients is unclear. L-carnitine has also been shown to reduce glycolysis and maintain a better pH in liquid stored platelets. This effect could result in extended platelet storage to 7 or 10 days. Based on such results, a role for L-carnitine as an additive to improve platelet quality in extended platelet storage is suggested.

Novel treatment modalities: new platelet preparations and substitutes.

Keywords: platelets; platelet substitutes; platelet haemostatic function; blood substitutes; platelet function

L-carnitine decreases glycolysis in liquid-stored platelets.

The platelet storage lesion is characterized metabolically by a pH decrease associated with lactic acid generation; a change in platelet morphology from discoid to spherical; a diminished response to in vitro challenge tests, such as the hypotonic shock response (HSR) and extent of shape change (ESC); increased surface P-selectin expression; and decreased in vivo recovery and survival. Altering storage conditions to improve these measures could allow for extension of the duration of in vitro storage. ABO-identical paired platelet concentrates were pooled and then equally divided into two plastic bags. Either L-carnitine (LC) or an equal volume of saline (control) was added to one container of each pair. Platelets were stored at 20 to 24 degrees C for 5 to 10 days or at 1 to 6 degrees C for 5 days at various concentrations of LC between 0.1 and 15 mM: At the end of storage, pH, glucose consumption, lactate generation, HSR, ESC, and surface P-selectin expression were measured. In different experiments, paired platelet concentrates were spiked with a Staphylococcus epidermidis suspension in the presence and absence of L-carnitine at a concentration of 5 mM: At 20 to 24 degrees C and concentrations of LC between 0.1 and 5 mM:, there was evidence of better pH preservation, less glucose consumption, and less lactate generation. Only with storage beyond 5 days was a difference present in either surface P-selectin expression or HSR. An L-carnitine concentration of 5 mM: appeared optimal. L-carnitine did not enhance the growth of bacteria after 7 to 8 days of storage. LC at 5 mM: may improve the quality of platelet concentrates that are stored beyond 5 days. There was no indication that LC at this concentration would promote bacterial growth. It may be a useful additive to platelet preservation.

Cryopreservation of human platelets with dimethyl sulfoxide: changes in biochemistry and cell function.

The shelf life of liquid-stored platelet concentrates is limited to 5 days. Therefore, much work has been carried out in an attempt to establish the optimum method for cryopreservation. Among the various cryoprotectants, dimethyl sulfoxide (DMSO) has been shown to be the most effective. However, DMSO-frozen platelets are characterized by a number of cell lesions. This report describes metabolic and functional changes that should give rise to some concern about the functional integrity of these cells. Single-donor platelet concentrates were frozen in liquid nitrogen by use of DMSO (5%). After thawing, the cells were washed and resuspended in autologous plasma. Before, during, and after the freezing process, samples for analysis of metabolic measures (e.g., pH; calcium, potassium, and lactate dehydrogenase concentrations; plasma complement factors) and functional measures (e.g., aggregometry, in vitro bleeding time, alpha-granule membrane protein-140 expression) were taken. Mean platelet volume increases during the deep-freezing process. Potassium, calcium, and lactate dehydrogenase are released from the intracellular space to the extracellular space. A strong activation of complement, which is mainly due to the addition of DMSO, is observed. Platelets become activated as indicated by the expression of alpha-granule membrane protein-140. Accordingly, decreased platelet function can be observed. DMSO-frozen platelets are characterized by several metabolic and functional changes. Although these cells have been shown to exert hemostatic effects in vivo, it is conceivable that those effects could be improved by further development of platelet-freezing techniques.

Membrane glycoproteins in cryopreserved platelets.

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.