α-Enolase From Injured Patients and Stored Packed Red Blood Cells Activates Pulmonary Endothelium and Serves as the First Event In a Two-Event Model of Neutrophil Cytotoxicity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3355-3355
Author(s):  
Nicole Tucker ◽  
Monika Dzieciatkowska ◽  
Kirk Hansen ◽  
Samina Khan ◽  
Marguerite Kelher ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Injury Severity ◽  
Conflicts Of Interest ◽  
Injured Patient ◽  
Post Injury ◽  
Packed Red Blood Cells ◽  
Severity Scores ◽  
Moonlighting Proteins ◽  
Injured Patients

Abstract Abstract 3355 A significant number of injured patients with intermediate injury severity scores (15<ISS<30) develop multiple organ failure (MOF), which clinically begins with acute lung injury (ALI). Transfusion of >6 units of stored PRBCs (≥28 days) is associated with the development of ALI/MOF on day 3 post injury (Am J Surg 178:502-4, 1999). The pro-inflammatory mediators, e.g. cytokines, responsible for MOF/ALI in the injured have remained elusive; therefore, we hypothesize that “moonlighting” proteins, which have defined intracellular function when released in the circulation, activate innate immunity and are etiologic in the development of ALI/MOF post-injury. Methods: Proteomics on the field blood (plasma) of 3 patients with blunt trauma who later developed ALI/MOF and the plasma from 3 units of packed red blood cells (PRBCs) on day 1 and day 42 were completed using 2-dimensional gel electrophoresis/mass spectroscopy (MALDI/TOF) with computer analyses of the resultant peptides. The proteins from whole cell lysates from Human pulmonary microvascular endothelial cells (HMVECs) were separted by SDS-PAGE, transferred to nitrocellulse and immunoblotted with antibodies to protease activated receptor-1 (PAR-1) and PAR-2. HMVECs were also incubated for 6 hours and 1) ICAM-1 was measured by flow cytometry, 2) isolated neutrophils (PMNs) were added allowed to settle and in selected wells PMN adherence to these activated HMVECs was measured by myeloperoxidase content in the lysate, or 3) after the PMNs settled, lysophosphatidylcholines (lyso-PCs) [4.5μM], lipids from stored platelets implicated in TRALI, were added and the number of viable HMVECs/mm2 were counted by microscopy. Results: HMVECs display immunoreactivity for both PAR-1 and PAR-2. Of the 243 proteins identified in the injured patients and the stored vs. fresh PRBCs, α-enolase increased by 10.8-fold and 4.4-fold respectively (p<.05 & p<.005). Both thrombin and α-enolase induced ICAM-1 expression in HMVECs (Table 1) which was inhibited (60±8%) by pre-treatment with the anti-protease leupeptin. α-Enolase also induced significant PMN adhesion vs. media control: Media: 3.1±1.5; α-enolase (50 μg/ml): 14.4±4.7; LPS: 35.5±0.7*. The α-enolase-activated HMVECs vs. buffer-treated lyso-PCs induced significant PMN-mediated cytotoxicity (Table 2). We conclude that α-enolase from the injured and stored but not fresh PRBCs causes pro-inflammatory activation of HMVECs resulting in PMN adherence and PMN cytotoxicity in a two-event in vitro model through activation of PARs receptors. Moonlighting proteins like the glycolytic lyase α-enolase may have unexpected pro-inflammatory activity, which predispose the injured patient to increased morbidity and mortality. Disclosures: No relevant conflicts of interest to declare.

Thrombin Activation of Protease-Activated Receptor-2 on Neutrophils Primes the Respiratory Burst.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1119-1119
Author(s):  
Ashley Bock ◽  
Marguerite Kelher ◽  
Samina Khan ◽  
Kirk Hansen ◽  
Monika Dzieciatkowska ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Respiratory Burst ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Gradient Centrifugation ◽  
Post Injury ◽  
Healthy Human ◽  
Whole Cell ◽  
Cell Lysates ◽  
Injured Patients

Abstract Abstract 1119 Thrombin is a multifunctional serine protease involved in hemostasis, fibrinolysis, and pro-inflammatory activation of innate immunity through stimulation of protease-activated receptors (PARs). Thrombin is considered the prototypical protease to activate PARs through cleavage of the N-terminal exodomain of the receptor, thereby unmasking a tethered ligand which activates it. Neutrophils (PMNs) are reported to express PAR-2; however, little is known about the changes in PMN physiology following protease activation of PAR-2. In addition, proteomic analyses (two dimensional gel electrophoresis and mass spectroscopy (MALDI-TOF)) of injured patients (blunt trauma) who developed acute lung injury (ALI) and multiple organ failure (MOF) have identified a number of serine proteases which accumulate post-injury. Moreover, these identical proteases were also found via proteomics in stored, but not fresh, packed red blood cells, which are used to resuscitate these injured patients. We hypothesize that thrombin primes PMNs through activation of PAR-2. Methods: PMNs were isolated from whole blood drawn from healthy human donors using dextran sedimentation, ficoll-hypaque gradient centrifugation and hypotonic lysis of red blood cells. The proteins from whole cell lysates were separated by SDS-polyacrylamide electrophoresis, transferred to nitrocellulose and immunoblotted with a monoclonal antibody to PAR-2. The PMNs were also incubated for 3–30 minutes at 37°C with thrombin [1-10 U/ml] followed by fMLP [1 μM] activation of the NADPH oxidase. Oxidase activity was measured by the superoxide dismutase-inhibitable reduction of cytochrome c at 550 nm. Results: PMNs express PAR-2 as detected by western blotting of whole cell lysates. Thrombin [1 U/ml] for 30 min, significantly primed the fMLP-activated respiratory burst: buffer control: 1.6 +/− 0.5 vs. 1 U/ml thrombin: 2.2 +/− 0.3* (*p<.05, n=5). Higher concentrations of thrombin or shorter incubation times did not induce PMN priming. Furthermore, pre-incubation with leupeptin [50 μM] and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) [250 μM], two effective protease inhibitors, abrogated the thrombin-induced PMN priming by 100±18%. We conclude that thrombin primes the PMN respiratory burst, which is dependent upon its protease activity. Because PMNs are the effector cell in ALI/MOF and priming agents have been linked to the development of clinical ALI and MOF, proteases that accumulate in the patient plasma post-injury or are transfused with stored PRBCs may be etiologic in post-injury ALI/MOF. Disclosures: No relevant conflicts of interest to declare.

Donor Variation In Biological Mediators During Storage Of Packed Red Blood Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3655-3655 ◽  
Author(s):  
Melinda M Dean ◽  
Luke D Samson ◽  
Kelly Rooks ◽  
Jesse Fryk ◽  
Shoma Baidya ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Immune Modulation ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Biological Response ◽  
Storage Lesion ◽  
Packed Red Blood Cells ◽  
Donor Variation ◽  
Time Point

Abstract Introduction During routine storage, packed red blood cells (PRBC) undergo numerous biochemical and biophysical changes collectively referred to as the “RBC storage lesion”. A number of factors reported to accumulate during the routine storage of PRBCs are hypothesized to mediate inflammatory cell responses and contribute to poor patient outcomes following transfusion. In addition, donor variability in red blood cell (RBC) characteristics and onset of the storage lesion has been reported. We investigated changes in levels of potential biological response modifies in the supernatant (SN) of PRBC relevant to storage, and, variance between donations. Methods Cytometric bead array was utilised to quantify a panel of 32 potential biological response modifiers (BRMs) in the SN of PRBC during storage. Potential BRMS were analysed in the SN of 8 leukodepleted PRBC units at weekly intervals (D2, D7, D14, D21, D28, D35, D42). The CBA panel was comprised of soluble(s) CD40 Ligand, sCD62E, sCD62L, sCD14, sCD54 (ICAM-1), sCD106 (VCAM-1), CXCL9, VEGF, Fractalkine (CX3CL1), IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF-α, MIP-1α, MIP-1β, IP-10, RANTES, sCD62P, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, IFN-α, IFN-γ, angiogenin, MCP-1. Storage related changes were analysed using ANOVA (95% CI). Donor variance was indicated by fold difference and range. “High” sub population of donations compared to remaining donations at each time point using Mann-Whitney (95% CI). Results Of the 32 potential BRMs studied, angiogenin, sCD14, sCD106 (VCAM-1), sCD62L, sCD62P, ICAM-1, IL-1α, IP-10, RANTES and IL-9 were consistently detected in all units throughout the time course. There was no evidence of a storage related increase in these biological mediators during storage of the PRBC, although angiogenin levels significantly declined during storage (P<0.001, ANOVA). Of particular interest, the concentrations of these nine biological mediators varied greatly between the individual PRBC units. ICAM-1, VCAM-1 and IL-1α concentrations each varied 10 fold between units (range 1000 – 10 000 pg/mL for each), sCD14 varied 5 fold (range 20 000 - 100 000 pg/mL), sCD62L varied 4.4 fold (range 9000 – 40 000 pg/mL), and sCD62P varied 6.5 fold (range 200 -1300 pg/ml). In addition, it was apparent that a sub population (3/8) of the units assessed consistently had the highest levels of ICAM-1, sCD106 (VCAM-1), sCD14, sCD62L, IL-1α, sCD62P and angiogenin. For sCD62P, in particular, this “high” sub population had significantly different levels of sCD62P at each time point compared to the other five units (P<0.05 at each time point). The remaining BRMs studied were at the limits of detection (<20 pg/mL) for every unit at each time point, and no storage related changes were evident. Conclusions There was minimal change in the BRMs studied relevant to storage duration of the PRBC units. The most notable differences in the levels of biological mediators present in PRBC SN were due to donor-to-donor variation. These data suggest high levels of BRMs and potential immune modulation in transfusion recipients may be the result of donor-associated differences rather than storage-associated differences in blood components. Disclosures: No relevant conflicts of interest to declare.

Incidents Related to the Transfusion By Hemosiderosis Notified to the National Haemovigilancy System: Results from Last Two YEARS in Our Hospital

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4745-4745
Author(s):  
Maria Almudena Garcia Ruiz ◽  
Romero Martinez Francisco Jose ◽  
Maria Pilar Garrido Collado ◽  
Estefania Morente Constantin ◽  
Manuel Jurado
Keyword(s):  
Iron Overload ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
World Health ◽  
Near Misses ◽  
Packed Red Blood Cells ◽  
History Of ◽  
Iron Overdose ◽  
Health Organization

Abstract OBJECTIVES According to the World Health Organization, "Haemovigilance is required to identify and prevent occurrence or recurrence of transfusion related unwanted events, to increase the safety, efficacy and efficiency of blood transfusion, covering all activities of the transfusion chain from donor to recipient." The system should include monitoring, identification, reporting, investigation and analysis of adverse events near-misses and reactions related to transfusion and manufacturing. Transfusion-dependent patients receive iron overdose in each transfusion: one packed red blood cells containing 200-250 mg iron (1 mg/ml), which accumulates gradually in different tissues. Transfusions of packed red blood cells in a volume ≥120 mL / kg can cause iron overload, which correlates with ferritin levels in serum equal to or greater than 1000 g/L. The iron overload may be detected after 10 to 20 transfused packed red blood cells, increasing the risk of morbidity and mortality. PATIENTS, MATERIAL AND METHODS We studied the hematological patients with iron overload in 2013 and 2014, analyzing theirs levels of ferritin. The database of the transfusion service was also used, as well as the transfusion history of patients who had received more than 10 packed red blood cells, correlating with greater than 1000 mg/L ferritin. In 2013, 57 cases transfused with packed red blood cells were reported: 32 men and 25 women between 20 and 87 years (average: 56). Results vary between 10 and 73 concentrates (average: 25). Posttransfusion ferritin levels exceeded 1.000 mg/L, with an average of 2869 mg/L. The accountability and severity was recorded as non-assessable. In 2014, 76 cases were reported between the ages of 24 and 83 years (average: 56 years), with 50 men and 26 women. The number of packed red blood cells transfused ranged from 10 to 130 (average: 31). The ferritin posttransfusion quantities ranged from 1.041 to 15.190 mg/L. Regarding accountability, 23 were grade 2, 43 were grade 1 and 10 were not assessable. The severity was recorded as non-assessable. Table. Cases reported in 2013 and 2014 Year Cases (nº) Average Packed red blood cells average per patient Posttransfusion ferritin levels 2.013 57 56 25 2.869 μg/L (average) 2.014 76 56 31 Values between 1041-15190 μg/L CONCLUSIONS It is very important to keep track of ferritin levels in polytransfused patients. In order to reduce the risk of hemosiderosis, it is essential the optimal use of component (safe, efficient and clinically effective) and that the transfusion is performed when the patient needs it. Chelation reduce morbidity in patients with transfusional dependency and iron overload. In the annual report of Andalusian Health Service, the incorporation of Haemovigilance system of notifications of post-transfusion hemosiderosis stands out, although only 4 hospitals shared the information, therefore working in this field is necessary. We believe it is essential to establish protocols to improve the reporting of incidents by hemosiderosis Haemovigilance System. Disclosures No relevant conflicts of interest to declare.

scholarly journals Blood on board: The development of a prehospital blood transfusion program in a Canadian helicopter emergency medical service

CJEM ◽  
2018 ◽  
Vol 21 (3) ◽  
pp. 365-373 ◽  
Author(s):  
Chase Krook ◽  
Domhnall O’Dochartaigh ◽  
Doug Martin ◽  
Zoë Piggott ◽  
Ryan Deedo ◽  
...  
Keyword(s):  
Blood Transfusion ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Helicopter Emergency Medical Service ◽  
Air Rescue ◽  
Packed Red Blood Cells ◽  
Standard Work ◽  
Emergency Medical ◽  
Set Up ◽  
Injured Patients

ABSTRACTObjectivePrehospital blood transfusion has been adopted by many civilian helicopter emergency medical services agencies, and early outcomes are positive. The Shock Trauma Air Rescue Society operates six bases in Western Canada and started a blood on board process in 2013 in Regina that has expanded to all bases. Two units of O negative packed red blood cells are carried on every mission. We describe the processes and standard work ensuring safe storage, administration, and stewardship of this important resource.MethodsThe packed red blood cells are stored in an inexpensive, reusable temperature controlled cooler at 1°C–6°C. Close collaboration with local transfusion services and adherence to Canadian transfusion standards contributes to safety and sustainability.ResultsFrom October 1, 2013 to October 10, 2017, the Shock Trauma Air Rescue Society administered blood to 431 patients. Of this total, 62.9% received blood carried on our aircraft. A total of 463 blood box units were administered, and the majority of patients (69.0%) received both units. Blood used in Calgary, Alberta was 100% traceable, and only 1.2% of total units dispensed was wasted. The vast majority of unused units were returned to circulation.ConclusionWe describe the process to set up and monitor a prehospital blood transfusion program. Our standard work and stewardship processes minimize wastage of blood while keeping it readily available for our critically ill and injured patients.

Apoptotic Microrna Profiling of Packed Red Blood Cells During Storage.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3145-3145
Author(s):  
Meganathan Kannan ◽  
Sandhya Kulkarni ◽  
Chintamani D Atreya
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Storage Period ◽  
Storage Condition ◽  
Packed Red Blood Cells ◽  
Cellular Expression ◽  
Underlying Mechanisms ◽  
Packed Rbcs

Abstract Abstract 3145 Poster Board III-82 Introduction During storage, RBCs undergo physiological changes often termed as storage lesions that adversely affect their survival in vivo, following transfusion. MicroRNAs (miRs), the negative regulators of cellular mRNAs, control cellular expression of genes relevant to differentiation and apoptosis via mRNA degradation or inhibition of translation. Recent reports indicate that matured red blood cells (RBC) contain diverse population of miRs in abundance. Understanding the role of miRs in RBC during storage would perhaps provide insights into the mechanisms associated with storage lesions. Methods In this study, we utilized a membrane-based array to obtain differential miR profiles of 52-apoptosis-associated miRs in packed RBCs during storage. The packed RBCs were obtained from the National Institutes of Health (NIH) blood bank and stored at appropriate storage condition (4-8°C) for up to 40 days. Samples were collected at days 0, 10, 20, 30 and day 40 and subjected to miR analysis. Our rationale is that since miRs are regulators of apoptosis, profiling of apoptosis associated miRs in packed RBCs during storage would provide the first step towards understanding the underlying mechanisms associated with storage lesions. Results Our miR analysis identified perturbation of six miRNAs during packed RBC storage. Two miRs remain at high levels throughout the RBC storage period studied while four miRs demonstrated an upward trend from day 0 to day 40 of storage. TarMir bioinformatics-based target gene identification for miR-96 identified CASPN1 mRNA as its target. The presence of CASPN1 mRNA was confirmed by RT-PCR. Although this observation tempts us to speculate that an interaction of CASPN1 with miR-96 in stored RBC is possible, further experimental verification of this interaction is warranted. Conclusion The differential microarray analysis presented here suggest that further refinement of miR profiling of stored red blood cells could be of value as predictive markers of ‘cellular status of RBC’ during storage. Future experimental analysis of selected mRNA-miR interactions in packed RBC during storage would provide insights into the mechanisms of storage lesions The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures No relevant conflicts of interest to declare.

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