Control Platelet Concentrates Research Articles

Assessment of metabolic and hemostatic profile of apheresis platelet concentrates: does the storage medium play a role?

The impact of pathogen reduction technology (PRT) on metabolic and haemostatic profile of treated platelets remains a subject of debate. Platelets Additive Solutions (PASs) are suggested as more appropriate storage medium compared to plasma. To investigate this in terms of zero heterogeneity PRT-treated and control apheresis platelet concentrates (PCs), collected from the same donors and stored in PAS and plasma respectively, were analyzed. In the first arm of the study six double dose-apheresis PCs were produced, split and stored in plasma, while in the second arm six split double dose-apheresis PCs from the same donors, were produced and stored in PAS. Control and PRT-treated PCs resulted in both arms. Metabolic and haemostatic markers were evaluated in all the examined groups on days 1, 3 and 5. A time dependent increased metabolism both in PAS and plasma-stored PCs was evident in PRT-treated PCs. However, the metabolic profile was better preserved in PCs stored in PAS, as higher pH (6.8 vs 6.5, p=0.007) and lower lactate levels (12.6 vs 17.8 mmol/L, p=0.009) were documented in PRT-treated PAS-PCs compared to plasma-PCs, on day 5. A time dependent decreased hemostatic capacity regardless the storage medium was evident in PRT-treated PCs, (PAS-PCs MCF, p=0.004 and plasma-PCs MCF, p=0.007). Similar results were obtained in control PCs. The use of PAS preserves the metabolic profile of PCs more adequately compared to plasma but has no effect on the haemostatic profile. The clinical relevance of these findings needs further investigation.

L-carnitine contributes to enhancement of viability and quality of platelet concentrates through changing the apoptotic and anti-apoptotic associated microRNAs

BackgroundMicro RNAs are known as the main regulator of messenger RNA translation in platelets and have a vital role in process of apoptosis during platelet storage. Our pervious study revealed that the expression of miR-145 and miR-326 changed significantly in platelets under maintenance conditions. This study aimed to evaluate the effect of L-carnitine (LC) as an additive to augment platelet quality by changing the microRNA expression. MethodsWe used ten platelet concentrate (PC) bags and divided each into two equal parts, LC- treated, and LC free PC. The expression of miR-145 and miR-326 were determined using real-time PCR. Moreover, we measured platelet count, platelet aggregation, platelet viability, and lactate dehydrogenase activity in all samples. ResultsThe miR-326 expression significantly increased during platelet storage with mean fold changes of 3.2 for the control and 2.5 for LC- treated PC. The mean fold changes in miR-145 expression was less in the control PC (0.52) compared to the LC- treated PC (0.79). Increased levels of platelet count, platelet aggregation, and platelet viability were found in the LC-treated compared to the untreated PC. ConclusionLC has a protective effect on platelet apoptosis, reduces the expression of apoptotic microRNA, and prevents the reduction of anti-apoptotic microRNA.

Two novel platelet biotinylation methods and their impact on stored platelet concentrates in a blood bank environment.

Storage of platelet concentrates (PCs) has an impact on platelet quality and possibly affects their functions after transfusion. The influence of processing and storage conditions of PCs on their in vivo function upon transfusion is unknown. One option for investigating this question is to implement an ex vivo labeling of human platelets, to analyze them after transfusion into heathy volunteers and/or patients. In this study, we developed two labeling methods employing biotin. Two methods of biotinylation were compared to a control (standard PC). The "Bio-Wash" process used washing steps to label all platelets within the PC; for the other method, "Bio-Direct," one fifth of the PC were directly labeled without washing steps. The control and the two biotinylated PCs were analyzed over 7 days of storage. Labeling efficiency, platelet counts, phenotypes, and functions, along with time and costs, were evaluated to select the best process. Both methods achieved a stable labeling through the storage, with similar platelet counts and metabolism in comparison to control PCs. Bio-Wash showed higher activation phenotype and lower aggregation response in comparison to the Bio-Direct method. The Bio-Direct was performed within 1.5 h versus 3 h for the Bio-Wash. However, the Bio-Direct required 12 mg of biotin instead of 8 mg for the other process. We set up two methods of biotinylation that can be easily implemented in a blood bank environment. The Bio-Direct process was preferred to the Bio-Wash because of its similarity, from a functional and phenotypic point of view, with standard PCs.

Transfusion of pathogen-reduced platelet components without leukoreduction.

BACKGROUNDLeukoreduction (LR) of platelet concentrate (PC) has evolved as the standard to mitigate risks of alloimmunization, clinical refractoriness, acute transfusion reactions (ATRs), and cytomegalovirus infection, but does not prevent transfusion‐associated graft‐versus‐host disease (TA‐GVHD). Amotosalen–ultraviolet A pathogen reduction (A‐PR) of PC reduces risk of transfusion‐transmitted infection and TA‐GVHD. In vitro data indicate that A‐PR effectively inactivates WBCs and infectious pathogens.STUDY DESIGN AND METHODSA sequential cohort study evaluated A‐PR without LR, gamma irradiation, and bacterial screening in hematopoietic stem cell transplant (HSCT) recipients. The first cohort received conventional PC (control) processed without LR, but with gamma irradiation and bacterial screening. The second cohort received A‐PR PC (test) processed without: LR, bacterial screening, or gamma irradiation. The primary efficacy outcome was the 1‐hour corrected count increment. The primary safety outcome was treatment‐emergent ATR. Secondary outcomes included clinical refractoriness, and 100‐day status for engraftment, TA‐GVHD, HSCT‐GVHD, infections, and mortality.RESULTSMean corrected count increment (× 103) of 33 test PC recipients was similar (18.9 ± 8.8 vs. 16.6 ± 8.4; p = 0.296) to that of 31 control PC recipients. Test recipients had a reduced, but nonsignificant, incidence of ATR (test = 9.1%, Control = 19.4%; p = 0.296). The frequencies of clinical refractoriness (0 of 33 vs. 4 of 31 patients) and refractory transfusions (6.6% vs. 19.3%) were lower in the test cohort (p = 0.05 and 0.02), respectively. No patient in either cohort had TA‐GVHD. Day 100 engraftment, HSCT‐GVHD, mortality, and infectious disease complications were similar between cohorts.CONCLUSIONSThis study indicated that A‐PR PC without LR, gamma irradiation, or bacterial screening is feasible for support of HSCT.

Differential Expression Profile of MicroRNAs During Prolonged Storage of Platelet Concentrates As a Quality Measurement Tool in Blood Banks.

Platelet concentrate (PC) is a key blood component, which even in good storage conditions, susceptible to cellular damage over time. Hence, blood banks discard unused PC bags after 5 days of storage. Biomarkers of PC quality are therefore highly sought after in blood bank governance. We used the data (Gene Expression Omnibus: GSE61856) generated with next-generation sequencing to examine the expression profiles of microRNAs (miRNAs) from PCs that were stored for 6 days in a blood bank, that is, 1 day longer than is normally stored PC. We identified the 14 most differentially expressed miRNAs by comparing a control PC on the first day of storage with the PCs on each of the subsequent 5 days of storage from day 1 to 6. In all, we identified nine miRNAs with the downregulated profile (miR-145-5p, miR-150-5p, miR-183-5p, miR-26a-5p, miR-331-3p, miR-338-5p, miR-451a, miR-501-3p, and miR-99b-5p) and five upregulated miRNAs (miR-1304-3p, miR-411-5p, miR-432-5p, miR-668-3p, and miR-939-5p). These miRNAs were validated by real-time quantitative PCR in 100 PC units. As each PC unit is composed of platelets of five individuals, the validation was thus performed in 500 individuals (250 men and 250 women, comprised 18-40 years old adults). The data were analyzed with hierarchical clustering and principal component analysis, which revealed the variation of mean relative expression and the instability of miRNAs half-life on the fourth day of PC storage, which coincides with time of onset of platelet storage lesions. These new observations can usefully inform future decision-making and governance in blood banks concerning PC quality.

A comprehensive proteomics study on platelet concentrates: Platelet proteome, storage time and Mirasol pathogen reduction technology

Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20–24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown in vitro that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to Mirasol PRT or PSL, and proves to be a methodology suitable to phenotype platelets in an unbiased manner, in various physiological contexts.

Mitochondrial damage-associated molecular patterns as potential proinflammatory mediators in post-platelet transfusion adverse effects.

Platelet concentrates (PCs) are the most common blood components eliciting nonhemolytic transfusion reactions (NHTRs), such as allergic transfusion reactions and febrile reactions. However, the precise mechanisms of NHTRs in PC transfusion remain largely unknown. Previous studies reported that mitochondria-derived damage-associated molecular patterns (DAMPs) could be important mediators of innate cell inflammation. Platelets (PLTs) represent a major reservoir of mitochondria in the blood circulation. The aim of this study was to determine the possible involvement of mitochondrial DAMPs in NHTRs. The amount of mitochondrial DAMPs was determined as an index of total copy numbers of mitochondrial DNA (mtDNA), including mtDNA itself and free mitochondria, using quantitative real-time polymerase chain reaction. To examine whether neutrophils, monocytes, and basophils were activated by mitochondrial DAMPs in vitro, an in vitro whole blood cell culture assay was performed. In blood components associated with NHTRs, the mean total mtDNA concentration was highest in PCs followed in order by fresh-frozen plasma and red blood cells. The amount of mtDNA in NHTR PCs was higher than that in control PCs without NHTRs. The mitochondrial DAMPs present in NHTR PCs was high enough to activate neutrophils, monocytes, and basophils, when costimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine or HLA antibodies. PLT-derived mitochondrial DAMPs are candidate risk factors for the onset of NHTRs.

Incidence of hypotension and acute hypotensive transfusion reactions following platelet concentrate transfusions.

Platelet concentrates (PCs) are associated with transfusion reactions involving hypotension, particularly bradykinin-mediated acute hypotensive transfusion reactions. This study aims to determine the incidence of hypotensive events and more specifically acute hypotensive transfusion reaction associated with PC transfusions. We also sought to ascertain whether these reactions are associated with elevated bradykinin levels. This is a prospective descriptive study of PCs administered at Sainte-Justine Hospital over 28 months. All PCs administered during this period were screened for hypotension through review of all transfusion-associated reaction reports (TARRs) sent to the blood bank. All residual PC bags were returned to the blood bank. TARRs associated with hypotension were reviewed by adjudicators that established the imputability of the PC transfusion to the reaction. Bradykinin levels were sampled in the first 168 PC bags returned to the blood bank. Levels were compared between PCs associated with hypotension and control PCs not associated with hypotension. A total of 3672 PC bags were returned to the blood bank; 25 PCs were associated with hypotension. Adjudicators ascertained that five hypotensive events were imputable to PCs of which one was an acute hypotensive transfusion reaction (incidence: 0·03%). Bradykinin level in the latter PC was 10 pg/ml, whereas levels were 226·2 ± 1252 pg/ml in the 143 control PCs. Our results show a low incidence of hypotension after PC transfusion. We identified only one acute hypotensive transfusion reaction. No correlation between bradykinin level and the occurrence of acute hypotensive reactions could be observed given that only one event was identified.

A hollow-fibre column system to effectively prepare washed platelets.

We developed a hollow-fibre column system specifically adapted to prepare washed platelet concentrates (WPCs). This study was performed to evaluate the efficacy of the hollow-fibre column system for preparing WPCs. First, the percentages of platelet (PLT) recovery and remaining plasma proteins were calculated by determining the PLT count, volume and plasma protein levels in both the prewash and postwash. Secondly, washed PLTs and unwashed control PLTs were stored for 5 days, and the changes during this 5-day storage of in vitro PLT characteristics were determined. The hollow-fibre column system effectively removed >98% of plasma in platelet concentrates (PCs), and the PLT recovery was 97% on an average. The CD62P-expression level on washed PLTs immediately after washing was approximately twofold higher than that on prewashed PLTs as well as on PLTs washed via manual methods or cell washing devices. Until day 5 during storage, PLT aggregability, hypotonic shock response and swirling scores of washed PLTs were not significantly different from those of the control PCs. Our novel hollow-fibre column system proved valuable in preparing washed PLTs with <2% of residual plasma proteins and high recovery of PLTs.

Hemostatic Function and Transfusion Efficacy of Apheresis Platelet Concentrates Treated with Gamma Irradiation in Use for Thrombocytopenic Patients

Background: During the transfusion of blood components, the transfer of allogeneic donor white blood cells (WBCs) can mediate transfusion-associated graft-versus-host disease (TA-GVHD). To minimize the reaction, exposure of blood products to gamma irradiation is currently the standard of care. The aim of our study was to evaluate and compare hemostatic function, transfusion efficacy, and safety of gamma-irradiated single-donor apheresis platelet concentrates (PCs) and of conventional non-irradiated PCs in patients with chemotherapy-induced thrombocytopenia. Methods: 20 double-dose single-donor leukoreduced PCs were split in two identical units; one was gamma-irradiated with 25 Gy (study arm A) and the other remains non-irradiated (study arm B). Both units were stored under equal conditions. Hematologic patients were randomly assigned to receive gamma-irradiated or conventional non-irradiated PCs. Hemostatic function was evaluated by thrombelastography (TEG). TEG measurements were taken pre transfusion and 1 and 24 h post transfusion. TEG profiles were measured, noting the time to initiate clotting (R), the angle of clot formation (α), and the maximum amplitude (clot strength (MA)). Whole blood samples were collected from these thrombocytopenic patients at 1 and 24 h for PLT count increments (CIs) and corrected count increments (CCIs) with assessments of transfusion efficacy. Time to next PLT transfusion, transfusion requirement of RBCs, active bleeding, and adverse events (AEs), were analyzed. Results: No differences could be found in hemostatic function parameters (MA, R, and α) between study arms A and B (all p values > 0.096) pre transfusion as well as 1 and 24 h post transfusion. No differences between study arms A and B were observed for mean (± standard deviation (SD)) 1-hour CCI (12.83 ± 6.33 vs. 11.59 ± 5.97) and 24-hour CCI (6.56 ± 4.10 vs. 5.76 ± 4.05). Mean 1-hour CI and 24-hour CI were not significantly different in both study arms (p = 0.254 and p = 0.242 respectively). Median time to the next PC transfusion after study PC was not significantly different between groups: (2.4 vs. 2.2 days, p = 0.767). No differences could be found in transfusion requirement of red blood cells (p = 0.744) between both study arms. There were also no regarding bleeding, adverse events, and acute transfusion reaction(s). Conclusions: This study confirms safety of gamma-irradiated PCs for treatment thrombocytopenia. Hemostatic function, transfusion efficacy, bleeding, and safety of single-donor apheresis PCs treated with gamma irradiation versus untreated control PCs are comparable.

Pathogen reduction treatment alters the immunomodulatory capacity of buffy coat–derived platelet concentrates

Storage of platelet concentrates (PCs) after Mirasol pathogen reduction technology (PRT) treatment changes platelet (PLT) surface marker expression and secretion of immunomodulatory factors. Given that PLTs are known to participate in immune function, PRT may alter the way PLTs interact with the immune cells of a recipient upon transfusion. As such, the aim of this study was to assess the effects of PRT treatment on the functional ability of PLTs to interact with peripheral blood mononuclear cells (PBMNCs). Buffy coat-derived PCs were pooled and split to obtain matched pairs. One unit was treated using the Mirasol PRT system, while the control PC remained untreated. After 5 days of storage, either the PLTs or the PLT supernatants from the PCs were cocultured with PBMNCs, with or without lipopolysaccharide (LPS). The immunomodulatory factors secreted into culture medium after coculture were examined. PRT-treated PLTs and PLT supernatant significantly increased the interleukin (IL)-8 concentration, which was manifested only in the presence of LPS. Conversely, PRT-treated PLTs secreted less soluble P-selectin (sCD62P) upon coculture with PBMNCs. PRT-treatment induced differential secretion of IL-8 and sCD62P during coculture, which may be attributed to either bioactive substances present in PLT supernatant or as a result of cell-cell interactions.

Effect of Mirasol pathogen reduction technology system on in vitro quality of MCS+ apheresis platelets

Reducing the risk of pathogen transmission to transfusion recipients is one of the great concerns in transfusion medicine. Important among the measures suggested to minimise pathogen transmission is pathogen reduction technology (PRT) systems. The present study examined the effects of Mirasol PRT system on MCS+ apheresis platelets in vitro quality measures during a seven-day storage period at 22°C. Statistical analysis indicated no significant difference in platelet concentrations between the control and treated platelet concentrates (PCs) during the storage period. Glucose and lactate levels were measured to determine metabolic activities of control and treated platelets. In both control and treated platelets, the amount of glucose consumed and lactate produced increased significantly with storage time, but glucose consumption and lactate production rates were significantly higher in treated platelets compared with control platelets. The mean pH of treated PCs was decreased at all time points relative to control PCs but remained within acceptable limits. The expression of P-selectin was also higher in Mirasol PRT treated platelets throughout the storage period, but differences were not statistically significant on Days 1 and 4. Finally, visual inspection of swirling indicated that Mirasol PRT treatment of platelets is associated with platelet shape change. Overall, our results show that MCS+ apheresis platelets treated with Mirasol PRT can preserve adequate in vitro properties for at least 5days of storage.

Proteomic analysis of platelets treated with gamma irradiation versus a commercial photochemical pathogen reduction technology

Several strategies are currently being tested to reduce the risk of pathogen transmission associated with platelet (PLT) transfusion. Within the framework of the Italian Platelet Technology Assessment Study, we investigated the variations of the protein profiles (proteomics) of apheresis PLT concentrates (PCs) upon treatment with riboflavin and ultraviolet (UV) light (Mirasol; 6.24 J/mL; 280-400 nm). Control, gamma-irradiated, and Mirasol-treated apheresis PCs were assayed on Days 1 and 5 of storage by means of gel-based analytical approaches (two-dimensional gel electrophoresis) and mass spectrometry-based identification of significant (p < 0.05 analysis of variance) differential proteins. Supernatants were then assayed for metabolism and oxidative stress-related metabolites through multiple reaction monitoring mass spectrometry. Only a handful of modifications could be observed in the PLT proteome profiles in response to the Mirasol treatment, which included proteins involved in oxidative stress responses, PLT metabolism, and activation. Results confirmed increased metabolic rate and oxidative stress in the supernatants of treated PLTs (both gamma irradiated and Mirasol treated). From this investigation, it emerges that, from a proteomics standpoint, gamma irradiation results in the acceleration of PLT storage lesions and the Mirasol treatment only moderately exacerbates these phenomena.

Preserved functional and biochemical characteristics of platelet components prepared with amotosalen and ultraviolet A for pathogen inactivation

Platelet concentrate (PC) functionality decreases during storage. This is referred to as the storage lesion. Pathogen inactivation may accelerate or induce lesions, potentially accounting for reduced viability. Our aim was to characterize functional and biochemical properties of platelets (PLTs) from photochemically treated buffy-coat PCs (PCT-PCs) compared to those from conventional PCs. Four PCT-PCs and four conventional PCs were stored for 6.5 days and PLT function and proteomic profiles were examined at various time points during storage. To evaluate their intrinsic properties, samples of stored PLTs were taken, washed, and suspended in Tyrode's buffer before testing. PLT counts and morphology were conserved although a slight increase in the PLT volume was observed after PCT. Glycoprotein (GP) IIbIIIa, IaIIa, and VI expression remained stable while GPIbα declined similarly in both types of PCs. A steep decrease (50%) in GPV occurred on Day 1.5 in PCT-PCs and Day 2.5 in control PCs. For both PCT- and control PCs, P-selectin expression and activated GPIIbIIIa remained low during storage. PCT- and control PCs were fully responsive to aggregation agonists up to Day 4.5 and exhibited similar perfusion functionality. Mitochondrial membrane potential and annexin A5 binding of PCT-PCs and control PCs were comparable. Two-dimensional differential in-gel electrophoresis and mass spectrometry profiles for 1882 protein spots revealed only three proteins selectively changed in PCT-PCs compared to control-PCs. Washed treated and untreated PCs have similar functional, morphologic, and proteomic characteristics provided that PLTs are suspended in an appropriate medium during testing.

Characteristics of the THERAFLEX UV-Platelets pathogen inactivation system – An update

Considerable progress has been made in the last decade in producing purer, safer, leucocyte and plasma reduced platelet concentrates (PC) with an extended shelf life. The development of different pathogen inactivation technologies (PIT) has made a substantial contribution to this trend. Preceding platelet PIT (INTERCEPT Blood System/Cerus Corporation, Concord, CA, USA; MIRASOL/Caridian BCT, Lakewood, CO, USA) are based on adding a photosensitive compound to PC. The mixture is then activated by UV light in the UVB and/or UVA spectral regions. A novel procedure, THERAFLEX UV-Platelets (MacoPharma, Mouvaux, France), was recently developed that uses short-wave ultraviolet light (UVC), without addition of any photoactive agent. This technology has proven to be highly effective in sterilising bacteria (the major cause of morbidity/mortality after platelet transfusion) as well as inactivating other transfusion transmitted DNA/RNA containing pathogens and residual leucocytes.Any PIT reflects a balance between the efficacy of pathogen inactivation and preservation of platelet quality and function. A broad spectrum of in vitro tests have become available for the assessment of platelet storage lesion (PSL), aiming to better predict clinical outcome and untoward effects of platelet therapy. Recent paired studies on the release of platelet-derived cytokines, as new platelet performance indicators, revealed a parallel increase in both THERAFLEX UV-treated and control PC throughout storage, supporting the notion that the bioavailability of platelet function is not grossly affected by UVC treatment. This is corroborated by some newer technologies for proteomic analysis, showing that the THERAFLEX UV-Platelets system results in limited disruption of integrin-regulating extracellular disulfide bonds and minimal protein alterations when compared to UVB and gamma irradiation. Moreover, standard in vitro parameters reflecting activation, metabolic activity and function of platelets are useful indicators of the overall performance of processing and storage and may be used as surrogate markers of platelet quality in vivo. However, there is some doubt as to what degree each marker alone or in combination reflects the true clinical outcome of transfused platelets. Therefore, an appropriate clinical programme has been initiated. The preclinical evaluation demonstrated tolerability and immunological safety of THERAFLEX UV-Platelets using an animal model. Additionally, the system has successfully completed two autologous Phase I trials on recovery and survival. Preliminary results suggest that the recovery and survival rates are consistent with other pathogen reduced platelet products that are licensed and in use. The method is currently under evaluation for safety and tolerability of UVC-treated platelets in healthy volunteers. Presently the THERAFLEX UV-Platelets system is the simplest and purest PIT easily adaptable to the existing blood bank setting. In the future, extension of the application range of the THERAFLEX UV-Platelets system is expected, in order to make this new technology compatible with a broad spectrum of collection and processing platforms, and with other blood products.

Platelet concentrates produced from whole blood using the Atreus processing system

The Atreus 2C+ system automates whole blood (WB) processing into a red cell concentrate, plasma and buffy coat (BC) suitable for platelet concentrate (PC) manufacture. This study compared the quality of PC made from BC using the Atreus, with those made by a manual method. WB was collected into Atreus disposables or standard bottom and top processing packs and held without active cooling for 26 h at 22 +/- 2 degrees C before processing, either with the Atreus, or using a centrifuge and press. BC were rested for 3 h and then 4 BC were pooled with one unit of plasma, mixed, centrifuged and pressed to make a pooled PC. The PC were analysed for quality markers to day 9 of storage. Platelet quality was good in both Atreus 2C+ derived PC and control units throughout storage. Metabolic markers (pH, ATP and HSR) and activation markers (CD62P, sCD62P, annexin V binding, microparticles, GP IIb/IIIa) did not differ between the Atreus and control units. Atreus-derived PC had significantly lower platelet yields (302 +/- 59 x 10(9) platelets/unit; mean +/- standard deviation, n = 8) than control PC (411 +/- 76 x 10(9) platelets/unit; P < 0.01), but met the UK guidelines for platelet yield. From these in vitro data, PC produced from buffy coats prepared using the Atreus appear suitable for clinical use, and WB may be held at ambient temperature overnight without the use of active cooling devices. Optimizing the secondary processing conditions to handle Atreus 2C+ derived BC may increase the platelet yield.

Small‐molecule complement inhibitors cannot prevent the development of the platelet storage lesion

Suppression of the platelet (PLT) storage lesion would maintain PLT quality over longer storage times. An increased storage period would greatly improve the ability of blood agencies and hospitals to manage PLT inventories and minimize product wastage. Activation of the complement system has been proposed to play a role in initiating or potentiating the PLT storage lesion. This study examines the effect of complement inhibition on the development of the PLT storage lesion. Leukofiltered PLT concentrates (PCs) were split into miniunits containing the complement inhibitors N-acetylaspartylglutamic acid (NAAGA) or compstatin, a control peptide, or saline. Samples were collected up to Day 11 of storage. Complement activation was monitored as C3a generation. PLT quality was assessed by morphology, CD62 and CD63 expression, fibrinogen binding, pH, mean PLT volume, annexin V binding, and PLT viability. Caspase-3 activity served as a measure of PLT apoptosis. At concentrations of NAAGA required to achieve approximately 50 percent complement inhibition, PLT activation, and caspase-3 activity were increased. Complement inhibition by compstatin was highly variable. Compstatin addition consistently resulted in a 37 to 55 percent inhibition of PLT caspase-3 activity, but PLT quality and viability were no different between compstatin PCs and control PCs over the storage time. Neither NAAGA nor compstatin provided complete inhibition of complement over the storage period. Addition of these small-peptide inhibitors to PCs did not slow PLT storage lesion development, in spite of the partial inhibition of caspase-3 activity in the compstatin-treated PCs.

In vitro evaluation of metabolic changes and residual platelet responsiveness in photochemical treated and gamma‐irradiated single‐donor platelet concentrates during long‐term storage

Photochemical treatment (PCT) prevents replication of pathogens in platelet concentrates (PCs) by cross-linking nucleic acids and thus affects all cells containing DNA or RNA. Fourteen double-dose single-donor PCs were divided into two study arms. The double-dose PCs were split in two identical units, PCT and conventional control PCs. Study Arm A consisted of seven PCT PCs with corresponding untreated controls, whereas Study Arm B consisted of seven PCT PCs with corresponding gamma-irradiated control. Metabolic changes and agonist-induced platelet (PLT) response were evaluated during storage for up to 12 days. Higher rate of PLT destruction, illustrated by reduced PLT content, increased lactate dehydrogenase levels, and higher CD61+ microparticle formation rate, were observed after PCT. Generally PCT accelerated metabolic changes in PCs and reduced agonist-induced (collagen or thrombin receptor activator peptide [TRAP]) aggregation responses. Flow cytometric analysis of CD62P and CD42b (GPIbalpha) expression showed higher spontaneous PLT activation in PCT PCs from 5 days of storage. Correspondingly, a reduced capacity for up regulation of CD62P expression and down regulation of CD42b was observed in PCT PLTs after stimulation by the agonists ADP or TRAP. Generally reduced in vitro PLT quality was observed after PCT during storage for up to 12 days, with marked reduction from 5 days of storage. Compared to conventional PCs, reduced agonist-induced aggregation and glycoprotein expression were observed after PCT during storage, corresponding to significantly higher level of spontaneous PLT activation in PCT PCs. Clinical studies of efficacy and safety of PCT PCs stored for more than 5 days are recommended.

Carbon Monoxide Atmosphere Allows Platelets To Retain Function and Hinder Pathogen Growth.

Platelets are the sole blood fraction which is currently preserved at room temperature and thus exposed to microorganisms, specifically bacterial, growth. Bacterial contamination in platelet concentrate (PC), in a range of up to 0.4%, is the major cause of morbidity and mortality in transfusion medicine. To extend the current limited shelf life of 5 days for platelets, fast screening methods have been developed. Alternatively, methods for inactivation of bacterial growth by DNA crosslinking are provided. The latter include treatment by psoralen or photodynamic inactivation such as riboflavin activated by irradiation. The drawback of these efficient methods which relates to large investment and unknown future side effects, calls for alternative preservation methods. In the current study we demonstrate an option to store PC under sealed anaerobic, carbon monoxide atmosphere, in which neutral pH is kept by bicarbonate (Cob). The procedure is simple and cost effective.Freshly isolated platelets (PC) were prepared by the Israel Central Blood Bank and stored in plastic flasks either allowing air transfer (control) or in gas non-transparent, Cob, at 22°C. Growth of inoculated bacteria in Cob stored PC was arrested immediately or after one log. Integrity of Cob platelets was better than control as assessed by leak of lactic dehydrogenase (LDH). Leak of LDH was lower as compared to control cells. P-selectin exposure on long term preserved platelets (days 6–9) was lower in Cob preserved platelets as compared to identical control PC. Phosophatydil serine (PS) measurement using flow cytometry indicated a large exposure in Cob platelets stored longer than 4 days, concomitant with consumption of all glucose, the energy source for anaerobic metabolism, in plasma. However, introduction of air resulted in a reversible reduction of the exposed PS. To better mimic in vivo viability of stored platelets, additional experiments were carried out using reconstituted blood (R-blood) prepared as follows: Platelets were removed from freshly drawn blood and stored PC were added to yield a concentration of ~ 350,000/μl. Aggregability of control and Cob R-blood were compared using a “paleletworks” kit (Helena company). Aggregability of stored PC was reduced with time. Nevertheless, aggregability of Cob platelets stored in R-blood was always significantly higher than its twin, air stored, sample. Adhesion of the stored platelets was estimated using Cone and Plate Analyzer (Diamed). No differences were found, within error, between air and Cob stored R-blood. In vivo survival of the stored platelets was assessed using a previously described rabbit model. Survival time declined with prolonged preservation in both control and COb stored platelets. No differences within error were observed between control and COb preserved platelets. These results indicate the option to use COb method for storage of platelets in a viable, pathogen reduced manner.

Soluble CD40 ligand accumulates in stored blood components, primes neutrophils through CD40, and is a potential cofactor in the development of transfusion-related acute lung injury

AbstractTransfusion-related acute lung injury (TRALI) is a form of posttransfusion acute pulmonary insufficiency that has been linked to the infusion of biologic response modifiers (BRMs), including antileukocyte antibodies and lipids. Soluble CD40 ligand (sCD40L) is a platelet-derived proinflammatory mediator that accumulates during platelet storage. We hypothesized that human polymorpho-nuclear leukocytes (PMNs) express CD40, CD40 ligation rapidly primes PMNs, and sCD40L induces PMN-mediated cytotoxicity of human pulmonary microvascular endothelial cells (HMVECs). Levels of sCD40L were measured in blood components and in platelet concentrates (PCs) implicated in TRALI or control PCs that did not elicit a transfusion reaction. All blood components contained higher levels of sCD40L than fresh plasma, with apheresis PCs evidencing the highest concentration of sCD40L followed by PCs from whole blood, whole blood, and packed red blood cells (PRBCs). PCs implicated in TRALI reactions contained significantly higher sCD40L levels than control PCs. PMNs express functional CD40 on the plasma membrane, and recombinant sCD40L (10 ng/mL-1 μg/mL) rapidly (5 minutes) primed the PMN oxidase. Soluble CD40L promoted PMN-mediated cytotoxicity of HMVECs as the second event in a 2-event in vitro model of TRALI. We concluded that sCD40L, which accumulates during blood component storage, has the capacity to activate adherent PMNs, causing endothelial damage and possibly TRALI in predisposed patients.